Substituted indoline derivatives as dengue viral replication inhibitors

ABSTRACT

The present invention concerns substituted indoline derivatives, methods to prevent or treat dengue viral infections by using said compounds and also relates to said compounds for use as a medicine, more preferably for use as a medicine to treat or prevent dengue viral infections. The present invention furthermore relates to pharmaceutical compositions or combination preparations of the compounds, to the compositions or preparations for use as a medicine, more preferably for the prevention or treatment of dengue viral infections. The invention also relates to processes for preparation of the compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage entry under 35 U.S.C. § 371 ofPCT International Patent Application No. PCT/EP2018/063029, filed May18, 2018, which claims priority to European Patent Application No.17172247.3, filed May 22, 2017, the contents of which are incorporatedherein by reference in their entirety.

The present invention relates to substituted indoline derivatives,methods to prevent or treat dengue viral infections by using saidcompounds and also relates to said compounds for use as a medicine, morepreferably for use as a medicine to treat or prevent dengue viralinfections. The present invention furthermore relates to pharmaceuticalcompositions or combination preparations of the compounds, to thecompositions or preparations for use as a medicine, more preferably forthe prevention or treatment of dengue viral infections. The inventionalso relates to processes for preparation of the compounds.

BACKGROUND OF THE INVENTION

Flaviviruses, which are transmitted by mosquitoes or ticks, causelife-threatening infections in man, such as encephalitis and hemorrhagicfever. Four distinct, but closely related serotypes of the flavivirusdengue are known, so-called DENV-1, -2, -3, and -4. Dengue is endemic inmost tropical and sub-tropical regions around the world, predominantlyin urban and semi-urban areas. According to the World HealthOrganization (WHO), 2.5 billion people of which 1 billion children areat risk of DENV infection (WHO, 2002). An estimated 50 to 100 millioncases of dengue fever [DF], half a million cases of severe denguedisease (i.e. dengue hemorrhagic fever [DHF] and dengue shock syndrome[DSS]), and more than 20,000 deaths occur worldwide each year. DHF hasbecome a leading cause of hospitalization and death amongst children inendemic regions. Altogether, dengue represents the most common cause ofarboviral disease. Because of recent large outbreaks in countriessituated in Latin America, South-East Asia and the Western Pacific(including Brazil, Puerto Rico, Venezuela, Cambodia, Indonesia, Vietnam,Thailand), numbers of dengue cases have risen dramatically over the pastyears. Not only is the number of dengue cases increasing as the diseaseis spreading to new areas, but the outbreaks tend to be more severe.

Following infection with another serotype, pre-existing heterologousantibodies form complexes with the newly infecting dengue virus serotypebut do not neutralize the pathogen. Instead, virus entry into cells isbelieved to be facilitated, resulting in uncontrolled virus replicationand higher peak viral titers. In both primary and secondary infections,higher viral titers are associated with more severe dengue disease.Since maternal antibodies can easily pass on to infants by breastfeeding, this might be one of the reasons that children are moreaffected by severe dengue disease than adults.

In locations with two or more serotypes circulating simultaneously, alsoreferred to as hyper endemic regions, the risk of serious dengue diseaseis significantly higher due to an increased risk of experiencing asecondary, more severe infection. Moreover, in a situation ofhyper-endemicity, the probability of the emergence of more virulentstrains is increased, which in turn augments the probability of denguehemorrhagic fever (DHF) or dengue shock syndrome.

The mosquitoes that carry dengue, including Aedes aegypti and Aedesalbopictus (tiger mosquito), are moving north on the globe. According tothe United States (US) Centers for Disease Control and Prevention (CDC),both mosquitoes are currently omnipresent in southern Texas. The spreadnorth of dengue-carrying mosquitoes is not confined to the US, but hasalso been observed in Europe.

Dengvaxia®, the dengue vaccine produced by Sanofi Pasteur was firstapproved in Mexico and has received in the meantime approval in morecountries. Nevertheless, the vaccine leaves considerable room forimprovement due to limited efficacy, especially against DENV-1 and -2,low efficacy in flavivirus-naïve subjects and the lengthy dosingschedule.

Despite these shortcomings, the vaccine is a game changer in endemicsettings as it will offer protection to a large part of the population,but likely not to very young infants, who bear the largest burden ofdengue. In addition, the dosing schedule and very limited efficacy inflavivirus-naïve subjects make it unsuitable and likely notworthwhile/cost-effective for travelers from non-endemic areas todengue-endemic areas. The above mentioned shortcomings of the denguevaccines are the reason why there is a need for a pre-exposureprophylactic dengue antiviral.

Furthermore, today, specific antiviral drugs for the treatment orprevention of dengue fever virus infection are not available. Clearly,there is still a great unmet medical need for therapeutics for theprevention or treatment of viral infections in animals, more inparticular in humans and especially for viral infections caused byflaviviruses, more in particular Dengue virus. Compounds with goodanti-viral potency, no or low levels of side-effects, a broad spectrumactivity against multiple Dengue virus serotypes, a low toxicity and/orgood pharmacokinetic or -dynamic properties are highly needed.

WO-2010/021878 discloses 2-phenylpyrrolidine and indoline derivatives ascold menthol receptor antagonists for treatment of inflammatory andcentral diseases. WO-2013/045516 discloses indole and indolinederivatives for use in the treatment of dengue viral infections.

The present invention now provides compounds, substituted indolinederivatives, which show high potent activity against all four (4)serotypes of the Dengue virus.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that at leastone of the above-mentioned problems can be solved by the currentcompounds of the invention.

The present invention provides compounds which have been shown topossess potent antiviral activity against all four (4) serotypescurrently known. The present invention furthermore demonstrates thatthese compounds efficiently inhibit proliferation of Dengue virus(DENV). Therefore, these compounds constitute a useful class of potentcompounds that can be used in the treatment and/or prevention of viralinfections in animals, mammals and humans, more specifically for thetreatment and/or prevention of infections with Dengue viruses.

The present invention furthermore relates to the use of such compoundsas medicines and to their use for the manufacture of medicaments fortreating and/or preventing viral infections, in particular with virusesbelonging to the family of the Dengue viruses in animals or mammals,more in particular in humans. The invention also relates to methods forthe preparation of all such compounds and to pharmaceutical compositionscomprising them in an effective amount.

The present invention also relates to a method of treatment orprevention of dengue viral infections in humans by the administration aneffective amount of one or more such compounds, or a pharmaceuticallyacceptable salt thereof optionally in combination with one or more othermedicines, like another antiviral agent, to a patient in need thereof.

The present invention concerns compounds of formula (I), including anystereo-chemically isomeric form thereof:

wherein

-   A is

wherein

-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    pentafluorosulfanyl, R⁵ is hydrogen, Z is carbon, and R⁶ is    hydrogen; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethyl, R⁵ is hydrogen, Z is carbon, and R⁶ is methyl; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethyl, R⁵ is fluoro, Z is carbon, and R⁶ is hydrogen; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is methyl; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethoxy, R⁵ is fluoro, Z is carbon, and R⁶ is hydrogen; or-   R¹ is fluoro, R² is methoxy, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen;    or-   R¹ is chloro, R² is hydrogen, R³ is deuterium, A is (a-1), R⁴ is    trifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen;    or-   R¹ is chloro, R² is —OCH₂CH₂OH, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen;    or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethyl, R⁵ is methoxy, Z is nitrogen, and R⁶ is absent; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-2), and R⁴ is    trifluoromethyl; or-   R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ is    trifluoromethylthio, R⁵ is hydrogen, Z is carbon, and R⁶ is    hydrogen;-   or a pharmaceutically acceptable salt, solvate or polymorph thereof.

A first group of compounds of formula (I) are those compounds of formula(I) wherein radical A is (a-1).

A second group of compounds of formula (I) are those compounds offormula (I) wherein radical A is (a-2).

In an alternative representation, the present invention relates to acompound having formula (I),

wherein

-   A is

wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt, solvate or polymorph thereof.

Part of the current invention is also a pharmaceutical compositioncomprising a compound mentioned above or a stereoisomeric form, apharmaceutically acceptable salt, solvate or polymorph thereof togetherwith one or more pharmaceutically acceptable excipients, diluents orcarriers.

Pharmaceutically acceptable salts of said compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Suitable base salts are formedfrom bases which form non-toxic salts.

The pharmaceutically acceptable acid salts as mentioned hereinabove aremeant to comprise the therapeutically active non-toxic acid additionsalt forms that the compounds of formula (I) are able to form. Thesepharmaceutically acceptable acid addition salts can conveniently beobtained by treating the base form with such appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butane-dioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic acid and the like acids.

The compounds of the invention may also exist in un-solvated andsolvated forms. The term “solvate” is used herein to describe amolecular complex comprising the compound of the invention and one ormore pharmaceutically acceptable solvent molecules, for example,ethanol.

The term “polymorph” refers to the ability of the compound of theinvention to exist in more than one form or crystal structure.

The compounds of the present invention may be administered ascrystalline or amorphous products. They may be obtained for example assolid plugs, powders, or films by methods such as precipitation,crystallization, freeze drying, spray drying, or evaporative drying.They may be administered alone or in combination with one or more othercompounds of the invention or in combination with one or more otherdrugs. Generally, they will be administered as a formulation inassociation with one or more pharmaceutically acceptable excipients. Theterm “excipient” is used herein to describe any ingredient other thanthe compound(s) of the invention. The choice of excipient dependslargely on factors such as the particular mode of administration, theeffect of the excipient on solubility and stability, and the nature ofthe dosage form.

The compounds of the present invention or any subgroup thereof may beformulated into various pharmaceutical forms for administrationpurposes. As appropriate compositions there may be cited allcompositions usually employed for systemically administering drugs. Toprepare the pharmaceutical compositions of this invention, an effectiveamount of the particular compound, optionally in addition salt form, asthe active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, for example, for oral or rectaladministration. For example, in preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed suchas, for example, water, glycols, oils, alcohols and the like in the caseof oral liquid preparations such as suspensions, syrups, elixirs,emulsions, and solutions; or solid carriers such as starches, sugars,kaolin, diluents, lubricants, binders, disintegrating agents and thelike in the case of powders, pills, capsules, and tablets. Because oftheir ease in administration, tablets and capsules represent the mostadvantageous oral dosage unit forms, in which case solid pharmaceuticalcarriers are obviously employed. Also included are solid formpreparations that can be converted, shortly before use, to liquid forms.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills, powderpackets, wafers, suppositories, injectable solutions or suspensions andthe like, and segregated multiples thereof.

Those of skill in the treatment of infectious diseases will be able todetermine the effective amount from the test results presentedhereinafter. In general it is contemplated that an effective dailyamount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferablyfrom 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate toadminister the required dose as two, three, four or more sub-doses atappropriate intervals throughout the day. Said sub-doses may beformulated as unit dosage forms, for example, containing 1 to 1000 mg,and in particular 5 to 200 mg of active ingredient per unit dosage form.

The exact dosage and frequency of administration depends on theparticular compound of the invention used, the particular conditionbeing treated, the severity of the condition being treated, the age,weight and general physical condition of the particular patient as wellas other medication the individual may be taking, as is well known tothose skilled in the art. Furthermore, it is evident that the effectiveamount may be lowered or increased depending on the response of thetreated subject and/or depending on the evaluation of the physicianprescribing the compounds of the instant invention. The effective amountranges mentioned above are therefore only guidelines and are notintended to limit the scope or use of the invention to any extent.

The present disclosure is also intended to include any isotopes of atomspresent in the compounds of the invention. For example, isotopes ofhydrogen include tritium and deuterium and isotopes of carbon includeC-13 and C-14.

As used herein, any chemical formula with bonds shown only as solidlines and not as solid wedged or hashed wedged bonds, or otherwiseindicated as having a particular configuration (e.g. R, S) around one ormore atoms, contemplates each possible stereoisomer, or mixture of twoor more stereoisomers.

Hereinbefore and hereinafter, the terms “compound of formula (I)” and“intermediates of synthesis of formula (I)” are meant to include thestereoisomers thereof and the tautomeric forms thereof.

The terms “stereoisomers”, “stereoisomeric forms” or “stereochemicallyisomeric forms” hereinbefore or hereinafter are used interchangeably.

The invention includes all stereoisomers of the compounds of theinvention either as a pure stereoisomer or as a mixture of two or morestereoisomers. Enantiomers are stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is aracemate or racemic mixture. Diastereomers (or diastereoisomers) arestereoisomers that are not enantiomers, i.e. they are not related asmirror images.

The term “stereoisomers” also includes any rotamers, also calledconformational isomers, the compounds of formula (I) may form.

Therefore, the invention includes enantiomers, diastereomers, racemates,E isomers, Z isomers, cis isomers, trans isomers, rotamers, and mixturesthereof, whenever chemically possible.

The meaning of all those terms, i.e. enantiomers, diastereomers,racemates, E isomers, Z isomers, cis isomers, trans isomers and mixturesthereof are known to the skilled person.

The absolute configuration is specified according to theCahn-Ingold-Prelog system. The configuration at an asymmetric atom isspecified by either R or S. Resolved stereoisomers whose absoluteconfiguration is not known can be designated by (+) or (−) depending onthe direction in which they rotate plane polarized light. For instance,resolved enantiomers whose absolute configuration is not known can bedesignated by (+) or (−) depending on the direction in which they rotateplane polarized light.

When a specific stereoisomer is identified, this means that saidstereoisomer is substantially free, i.e. associated with less than 50%,preferably less than 20%, more preferably less than 10%, even morepreferably less than 5%, in particular less than 2% and most preferablyless than 1%, of the other stereoisomers. Thus, when a compound offormula (I) is for instance specified as (R), this means that thecompound is substantially free of the (S) isomer.

Some of the compounds according to formula (I) may also exist in theirtautomeric form. Such forms in so far as they may exist, although notexplicitly indicated in the above formula (I) are intended to beincluded within the scope of the present invention.

The compounds of formula (I) of the present invention all have at leastone asymmetric carbon atom as indicated in the figure below by thecarbon atom labelled with *:

Due to the presence of said chiral center, a “compound of formula (I)”can be the (R)-enantiomer, the (S)-enantiomer, the racemic form, or anypossible combination of the two individual enantiomers in any ratio.When the absolute (R)- or (S)-configuration of an enantiomer is notknown, this enantiomer can also be identified by indicating whether theenantiomer is dextrorotatory (+)- or levorotatory (−)- after measuringthe specific optical rotation of said particular enantiomer.

In an aspect the present invention relates to a first group of compoundof formula (I) wherein the compounds of formula (I) have the (+)specific rotation.

In a further aspect the present invention relates to a second ground ofcompounds of formula (I) wherein the compounds of formula (I) have the(−) specific rotation.

EXAMPLES

LC/MS Methods

The High Performance Liquid Chromatography (HPLC) measurement wasperformed using a LC pump, a diode-array (DAD) or a UV detector and acolumn as specified in the respective methods. If necessary, additionaldetectors were included (see table of methods below).

Flow from the column was brought to the Mass Spectrometer (MS) which wasconfigured with an atmospheric pressure ion source. It is within theknowledge of the skilled person to set the tune parameters (e.g.scanning range, dwell time . . . ) in order to obtain ions allowing theidentification of the compound's nominal monoisotopic molecular weight(MW). Data acquisition was performed with appropriate software.

Compounds are described by their experimental retention times (R_(t))and ions. If not specified differently in the table of data, thereported molecular ion corresponds to the [M+H]⁺ (protonated molecule)and/or [M−H]⁻ (deprotonated molecule). In case the compound was notdirectly ionizable the type of adduct is specified (i.e. [M+NH₄]⁺,[M+HCOO]⁻, etc. . . . ). For molecules with multiple isotopic patterns(Br, Cl), the reported value is the one obtained for the lowest isotopemass. All results were obtained with experimental uncertainties that arecommonly associated with the method used.

Hereinafter, “SQD” means Single Quadrupole Detector, “MSD” MassSelective Detector, “RT” room temperature, “BEH” bridgedethylsiloxane/silica hybrid, “DAD” Diode Array Detector, “HSS” HighStrength silica.

LC/MS Method codes (Flow expressed in mL/min; column temperature (T) in° C.; Run time in minutes).

Run Method code Instrument Column Mobile phase Gradient$\frac{Flow}{{Col}\mspace{14mu} T}$ time (min) LC-A Waters: Acquity ®Waters: BEH ® C18 (1.7 μm, 2.1 × A: 95% CH₃COONH₄ 84.2% A for 0.49 min,to 10.5% A in 2.18 min,$\frac{0.343\mspace{14mu}{{mL}/\min}}{40{^\circ}\mspace{14mu}{C.}}$ 6.2UPLC ®-DAD- 100 mm) 7 mM/5% held for 1.94 min, back Quattro CH₃CN, to84.2% A in 0.73 min, Micro ™ B: CH₃CN held for 0.73 min. LC-B Waters:Acquity ® H- Waters: BEH ® C18 (1.7 μm, 2.1 × A: 95% CH₃COONH₄ 84.2%A/15.8% B to 10.5% A in 2.18 min,$\frac{0.343\mspace{14mu}{{mL}/\min}}{40{^\circ}\mspace{14mu}{C.}}$ 6.1Class-DAD 100 mm 7 mM/5% held for 1.96 min, back and SQD2TM CH₃CN, to84.2% A/15.8% B in B: CH₃CN 0.73 min, held for 0.49 min. LC-C Waters:Acquity ® Waters: BEH C18 (1.7 μm, 2.1 × A: 10 mM CH₃COONH₄ From 95% Ato 5% A in 1.3 min, held for 0.7$\frac{0.8\mspace{14mu}{{mL}/\min}}{55{^\circ}\mspace{14mu}{C.}}$ 2  UPLC ®-DAD- 50 mm) in 95% H₂O + min. SQD 5% CH₃CN B: CH₃CN LC-D Waters:Acquity ® Waters: HSS T3 (1.8 μm, 2.1 × A: 10 mM CH₃COONH₄ From 100% Ato 5% A in 2.10 min,$\frac{0.7\mspace{14mu}{{mL}/\min}}{55{^\circ}\mspace{14mu}{C.}}$ 3.5UPLC ®-DAD- 100 mm) in 95% H₂O + to 0% A in 0.90 min, to SQD 5% CH₃CN 5%A in 0.5 min B: CH₃CN

SFC/MS Methods

The SFC measurement was performed using an Analytical Supercriticalfluid chromatography (SFC) system composed by a binary pump fordelivering carbon dioxide (CO2) and modifier, an autosampler, a columnoven, a diode array detector equipped with a high-pressure flow cellstanding up to 400 bars. If configured with a Mass Spectrometer (MS) theflow from the column was brought to the (MS). It is within the knowledgeof the skilled person to set the tune parameters (e.g. scanning range,dwell time . . . ) in order to obtain ions allowing the identificationof the compound's nominal monoisotopic molecular weight (MW).

Data acquisition was performed with appropriate software. AnalyticalSFC/MS Methods (Flow expressed in mL/min; column temperature (T) in °C.; Run time in minutes, Backpressure (BPR) in bars.

Method code column mobile phase gradient$\frac{Flow}{{Col}\mspace{14mu} T}$$\frac{{Run}\mspace{14mu}{time}}{BPR}$ SFC-A Daicel Chiralpak ® A: CO₂B: EtOH (+0.2% 10%-50% B in 6 min, $\frac{2.5}{40}$ $\frac{9.5}{110}$AS3 column iPrNH₂ + hold 3.5 min (3.0 μm, 150 × 4.6 mm) 3% H₂O) SFC-BDaicel Chiralpak ® OD3 column A: CO₂ B: EtOH (+0.2% 10%-50% B in 6 min,$\frac{2.5}{40}$ $\frac{9.5}{110}$ (3.0 μm, 150 × 4.6 mm) iPrNH₂) hold3.5 min SFC-C Daicel Chiralpak ® AS3 column A: CO₂ B: EtOH (+0.2%10%-50% B in 6 min, $\frac{2.5}{40}$ $\frac{9.5}{110}$ (3.0 μm, 150 ×4.6 mm) iPrNH₂) hold 3.5 min SFC-D Daicel Chiralcel ® OD-3 column A: CO₂B: iPrOH (+0.3% 40% B hold 3 min $\frac{3.5}{35}$ $\frac{3}{105}$ (3 μm,100 × 4.6 mm) iPrNH₂) SFC-E Whelk ®-O-(R,R) column A: CO₂ B: EtOH (+0.2%10%-50% B in 6 min, $\frac{2.5}{40}$ $\frac{9.5}{110}$ (5.0 μm, 250 ×4.6 mm) iPrNH₂) hold 3.5 min SFC-F Daicel Chiralpak ® ID3 column A: CO₂B: EtOH (+0.2% 10%-50% B in 6 min, $\frac{2.5}{40}$ $\frac{9.5}{110}$(3.0 μm, 150 × 4.6 mm) iPrNH₂) hold 3.5 min SFC-G Regis Whelk O1, S,Scolumn A: CO₂ B: MeOH 40% B hold 3 min, $\frac{3.5}{35}$ $\frac{3}{103}$(3 μm, 100 × 4.6 mm) SFC-H Daicel Chiralcel ® OD-3 column A: CO₂ B: MeO40% B hold 3 min, $\frac{3.5}{35}$ $\frac{3}{103}$ (3 μm, 100 × 4.6 mm)SFC-I Daicel Chiralcel ® OD-3 column A: CO₂ B: MeOH 30% B hold 3 min,$\frac{3.5}{35}$ $\frac{3}{103}$ (3 μm, 100 × 4.6 mm)

Melting Points Values are either peak values or melt ranges, and areobtained with experimental uncertainties that are commonly associatedwith this analytical method.

DSC823e (Indicated as DSC)

For a number of compounds, melting points were determined with a DSC823e(Mettler-Toledo). Melting points were measured with a temperaturegradient of 10° C./minute. Maximum temperature was 300° C.

Optical Rotations:

Optical rotations were measured on a Perkin-Elmer 341 polarimeter with asodium lamp and reported as follows: [α]° (λ, c g/100 ml, solvent, ToC).[α]_(λ) ^(T)=(100a)/(l×c): where l is the path length in dm and c is theconcentration in g/100 ml for a sample at a temperature T (° C.) and awavelength A (in nm). If the wavelength of light used is 589 nm (thesodium D line), then the symbol D might be used instead. The sign of therotation (+ or −) should always be given. When using this equation theconcentration and solvent are always provided in parentheses after therotation. The rotation is reported using degrees and no units ofconcentration are given (it is assumed to be g/100 ml).

Abbreviations used in experimental part (M + H)⁺ MH⁺ protonatedmolecular ion iPrNH₂ isopropylamine aq. aqueous iPrOH 2-propanol Boctert-butyloxycarbonyl K₂CO₃ potassium carbonate Boc₂O di-tert-butyldicarbonate KNO₃ potassium nitrate br broad LiAlH₄ lithium aluminiumhydride CH₃CN acetonitrile m/z mass-to-charge ratio CHCl₃ chloroform Memethyl CH₂Cl₂ dichloromethane MeOH methanol CH₃OH methanol MgSO₄magnesium sulfate CO₂ carbon dioxide min minute(s) CsCO₃ cesiumcarbonate MTBE methyl-tert-butylether d doublet N₂ nitrogen DCMdichloromethane Na₂CO₃ sodium carbonate DIEA diisopropylethylamineNa₂SO₄ sodium sulfate DIPE diisopropyl ether NaBH₄ sodium borohydrideDMA dimethylacetamide NaCl sodium chloride DMAP 4-dimethylaminopyridineNaHCO₃ sodium bicarbonate DME 1,2-dimethoxyethane NaOH sodium hydroxideDMF dimethylformamide NH₄Cl ammonium chloride DMSO dimethyl sulfoxideNH₄HCO₃ ammonium bicarbonate EDCl 1-ethyl-3-(3-dimethylamino- NMPN-methylpyrrolidon propyl)carbodiimide eq. equivalent q quartet Et₂Odiethyl ether rt or RT room temperature Et₃N triethylamine SEMCl2-(trimethylsilyl)ethoxymethyl chloride EtOAc ethyl acetate s singletEtOH ethanol t triplet H₂ hydrogen tBuOK potassium tert-butanolaat HNO₃nitric acid TEA H₂O water TFA H₂SO₄ sulfuric acid THF HATUO-7-aza-H-benzotriazol-1- 2-Me—THF 2-methyltetrahydrofuranyl)-N,N,N′,N′-tetramethyl- triethylamine uronium hexafluorophosphate-trifluoroacetic acid CAS [148893-10-1] tetrahydrofuran HCl hydrochloricacid TMSCl trimethylsilyl chloride HPLC high performance liquid TMSCF₃trifluoromethyltrimethylsilane chromatography

Example 1: Synthesis of4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 1) and Chiral Separation into Enantiomers 1A and 1B

Synthesis of Intermediate 1a

To a mechanically stirred solution of tert-butyl 4-bromobutanoate [CAS110661-91-1] (42.3 g, 0.19 mol) in DMF (600 mL) was added in portions asolid mixture of 3-amino-5-methoxyphenol [CAS 162155-27-3] (26.4 g, 0.19mol) and Cs₂CO₃ (123.6 g, 0.379 mol). The reaction was stirred at 60° C.for 65 h, and allowed to reach room temperature. The mixture was pouredout into H₂O (2.5 L). The product was extracted with Et₂O (2 times). Thecombined organic layers were washed with brine, dried over MgSO₄ andfiltered. The solvent was evaporated under reduced pressure, andco-evaporated with toluene. The residue was purified via Normal PhaseHPLC (Stationary phase: silica gel 60A 25-40 μm (Merck), mobile phase:gradient from 20% EtOAc, 80% heptane to 60% EtOAc, 40% heptane) yieldingtert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate 1a (27 g).

Synthesis of Intermediate 1b

At 0° C., BH₃—Pyridine (1.46 mL, 14.5 mmol) was added slowly to asolution of 6-(pentafluoro-λ⁶-sulfanyl)-1H-indole [CAS 1379811-84-3](1.0 g, 4.11 mmol) in EtOH (8.5 mL). 5N HCl (7 mL) was slowly added. Themixture was stirred at 0° C. for 2 h and allowed to gradually warm toroom temperature while stirring overnight. After cooling to 0° C.(ice-bath), 50% NaOH (2 mL) was added dropwise and stirring wascontinued for 15 min. Water (50 mL) was added and the product wasextracted with Et₂O/EtOAc 2/1. The organic layer was separated, driedover MgSO₄, filtered and the solvent was evaporated under reducedpressure. The residue was purified by flash chromatography on silica gel(25 g) using a gradient of heptane/CH₂Cl₂ 100/0 to 0/100. The productfractions were combined and evaporated under reduced pressure. Theresidue was dried under vacuum at 45° C. to give6-(pentafluoro-λ⁶-sulfanyl)indoline 1b (328 mg).

Synthesis of Intermediate 1c

A mixture of give 6-(pentafluoro-l⁶-sulfanyl)indoline 1b (328 mg, 1.34mmol), 2-(4-chlorophenyl)acetic acid [CAS 1878-66-6] (228 mg, 1.34mmol), HATU (778 mg, 2.0 mmol) and diisopropylethylamine (663 μL, 4.0mmol) in CH₃CN (15 mL) was stirred at room temperature for 65 h. Thesolvent was evaporated under reduced pressure. The residue was dissolvedin 2-Me-THF (50 mL) and washed with 1N HCl (25 mL) and brine. Theorganic layer was separated, dried over MgSO₄, filtered, and the solventwas evaporated under reduced pressure. The residue was purified by flashchromatography on silica gel (12 g) using a gradient of heptane/EtOAc100/0 to 0/100. The desired fractions were combined and evaporated underreduced pressure. The product was crystallized from CH₂Cl₂/EtOAc,filtered off, washed (3×) with EtOAc, and dried under vacuum at 45° C.to provide2-(4-chlorophenyl)-1-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)-ethanone1c (209 mg). The filtrate was evaporated under reduced pressure. Theresidue was stirred up in Et₂O (2 mL), filtered off, washed (3×) withEt₂O, and dried at under vacuum at 45° C. to provide a second crop ofintermediate 1c (155 mg).

Synthesis of Intermediate 1d

At −70° C., under a N₂ flow, LiHMDS 1M in THF (1.78 mL, 1.78 mmol) wasadded dropwise to a mixture of2-(4-chlorophenyl)-1-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethanone1c (354 mg, 0.89 mmol) in 2-Me-THF (35 mL) and the mixture was kept at−70° C. for 30 min. TMSCl (182 μL, 1.42 mmol) was added dropwise. Themixture was stirred for 30 min at −70° C. and a solution ofN-bromosuccinimide (198 mg, 1.11 mmol) in a solvent mixture of THF (1.5mL) and 2-Me-THF (5 mL) was added dropwise. After stirring for 1 h at−78° C., the reaction was quenched with a saturated aqueous solution ofNH₄Cl (50 mL). The cooling bath was removed and the reaction mixture wasstirred for 50 min. Water (10 mL) was added and the organic layer wasseparated, dried over MgSO₄, filtered and the solvent was evaporatedunder reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethanone1d (424 mg), which was used as such in the next step.

Synthesis of Compound 1 and Chiral Separation into Enantiomers 1A and 1B

A mixture of2-bromo-2-(4-chlorophenyl)-1-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethanone1d (424 mg, 0.89 mmol), tert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate1a (260 mg, 0.92 mmol) and diisopropylethylamine (306 μL, 1.78 mmol) inCH₃CN (30 mL) was stirred at 60° C. for 18 h. The reaction mixture wasallowed to reach room temperature, and poured out into stirring water(150 mL). The product was extracted (2×) with Et₂O. The combined organiclayers were washed with brine, dried over MgSO₄, filtered, and thesolvent was evaporated under reduced pressure. The residue was purifiedby flash chromatography on silica gel (40 g) using a gradient ofheptane/EtOAc/EtOH 100/0/0 to 40/45/15). The desired fractions werecombined, evaporated under reduced pressure, and co-evaporated withdioxane.

The residue (602 mg, containing 58% of intermediate 1e) was mixed with4M HCl in dioxane (4 mL) and the mixture was stirred at room temperaturefor 5 h. The solids were filtered off, washed with dioxane (3×) and Et₂O(2×), and dried under vacuum at 45° C. to provide crude4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 1, 309 mg). An analytical sample (60 mg) of racemicCompound 1 was further purified via preparative HPLC (Stationary phase:RP XBridge® Prep C18 OBD—10 μm, 30×150 mm, mobile phase: 0.25% NH₄HCO₃solution in water, CH₃CN). The pure fractions were combined and theorganic volatiles were evaporated under reduced pressure. The remainingaqueous solution was co-evaporated under reduced pressure with o-xylene.The residue was dissolved in a solvent mixture of CH₃CN and water,evaporated under reduced pressure and co-evaporated with dioxane. Theresidue was lyophilized from a solvent mixture of CH₃CN (2 mL) and water(0.8 mL) to provide pure4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(pentafluoro-λ⁶-sulfanyl)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 1, 40 mg) as a powder.

The enantiomers of Compound 1 (249 mg) were separated via preparativechiral SFC (Stationary phase: Chiralpak® Diacel AD 20×250 mm, mobilephase: CO₂, EtOH+0.4% iPrNH₂). The product fractions of the first elutedenantiomer were combined, evaporated under reduced pressure andco-evaporated with MeOH. The residue was stirred up in water (3.5 mL)and MeOH (1 mL), the solids were filtered off, washed (3×) withwater/MeOH 4/1, and dried under vacuum at 45° C. to provide Enantiomer1A (41 mg). The product fractions of the second eluted enantiomer werecombined, evaporated under reduced pressure and co-evaporated with MeOH.The residue was stirred up in water (3 mL) and MeOH (0.6 mL), the solidswere filtered off, washed (3×) with water/MeOH 4/1, and dried undervacuum at 45° C. to provide Enantiomer 1B (48 mg).

Compound 1:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.86 (quin, J=6.8 Hz, 2H) 2.33 (t, J=7.3Hz, 2H) 3.12-3.25 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.5 Hz, 2H) 3.99-4.13(m, 1H) 4.47-4.59 (m, 1H) 5.57 (d, J=8.6 Hz, 1H) 5.76 (t, J=2.1 Hz, 1H)5.91-5.96 (m, 2H) 6.45 (d, J=8.6 Hz, 1H) 7.39-7.50 (m, 3H) 7.51-7.62 (m,3H) 8.58 (d, J=2.0 Hz, 1H) 12.12 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.09 min, MH⁺ 621

Enantiomer 1A:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.85 (quin, J=6.8 Hz, 2H) 2.26 (br t,J=6.8 Hz, 2H) 3.15-3.25 (m, 2H) 3.61 (s, 3H) 3.84 (br t, J=6.4 Hz, 2H)4.02-4.12 (m, 1H) 4.48-4.60 (m, 1H) 5.59 (d, J=8.8 Hz, 1H) 5.76 (t,J=2.0 Hz, 1H) 5.93 (t, J=2.0 Hz, 1H) 5.96 (t, J=2.0 Hz, 1H) 6.47 (d,J=8.4 Hz, 1H) 7.42-7.47 (m, 3H) 7.53-7.59 (m, 3H) 8.58 (d, J=2.2 Hz, 1H)

LC/MS (method LC-D): R_(t) 1.99 min, MH⁺ 621

[α]_(D) ²⁰: −44.6° (c 0.28, DMF)

Chiral SFC (method SFC-A): R_(t) 3.54 min, MH⁺ 621 chiral purity 97.9%.

Enantiomer 1B:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.86 (quin, J=6.8 Hz, 2H) 2.33 (t, J=7.3Hz, 2H) 3.14-3.29 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.4 Hz, 2H) 4.01-4.11(m, 1H) 4.48-4.58 (m, 1H) 5.58 (d, J=9.1 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H)5.93 (t, J=1.8 Hz, 1H) 5.94-5.96 (m, 1H) 6.48 (d, J=9.1 Hz, 1H)7.41-7.48 (m, 3H) 7.52-7.61 (m, 3H) 8.58 (d, J=2.2 Hz, 1H) 12.13 (br s,1H)

LC/MS (method LC-D): R_(t) 1.98 min, MH⁺ 621

[α]_(D) ²⁰: +46.0° (c 0.265, DMF)

Chiral SFC (method SFC-A): R_(t) 3.82 min, MH⁺ 621 chiral purity 99.0%.

Example 2: Synthesis of4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 2) and Chiral Separation into Enantiomers 2A and 2B

Synthesis of Intermediate 2a

Pd/C (10%) (1.18 g) was added to a solution of1-benzyl-4-methyl-6-(trifluoromethyl)indoline [CAS 1156512-79-6] (11.8g, 40.5 mmol) in AcOH (11.8 mL) and MeOH (118 mL). The reaction wasstirred at room temperature for 12 h under H₂ atmosphere. The mixturewas filtered through a pad of Celite® and concentrated under reducedpressure. The residue was taken up with CH₂Cl₂, washed with water,brine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gel(heptane/EtOAc 9/1). The pure fractions were combined and the solventwas evaporated to dryness to give 8.2 g of4-methyl-6-(trifluoromethyl)indoline 2a.

Synthesis of Intermediate 2b

tert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate 1a (2.94 g, 10.5 mmol)was added to solution of methyl 2-bromo-2-(4-chlorophenyl)acetate [CAS24091-92-7](2.51 g, 9.53 mmol) in CH₃CN (200 mL). Diisopropylethylamine(2.46 mL, 14.3 mmol) was added and the reaction mixture was stirred at80° C. overnight. The solvent was evaporated under reduced pressure. Theresidue was dissolved in CH₂Cl₂ and washed with 1N HCl. The organiclayer was washed with water, dried over MgSO₄, filtered, and evaporatedto dryness under reduced pressure. The residue was purified by columnchromatography on silica gel (100 g) using a gradient of EtOAc:EtOH(3:1)/heptane 0/100 to 50/50. The product fractions were combined, andevaporated under reduced pressure and the residue was dried under vacuumat 50° C. to provide tert-butyl4-(3-((1-(4-chlorophenyl)-2-methoxy-2-oxoethyl)amino)-5-methoxyphenoxy)butanoateas2b (3.74 g) as a yellow oil.

Synthesis of Intermediate 2c

Lithium hydroxide (336 mg, 14.0 mmol) was added to a solution oftert-butyl4-(3-((1-(4-chlorophenyl)-2-methoxy-2-oxoethyl)amino)-5-methoxyphenoxy)butanoateas2b (3.74 g, 7.02 mmol) in a solvent mixture of water (25 mL), MeOH (25mL) and THF (75 mL) and the reaction mixture was stirred at roomtemperature for 5 h. Saturated aqueous NH₄Cl (50 mL) was added and theorganic volatiles were evaporated under reduced pressure. The residualaqueous solution was acidified with 1N HCl to pH 2 and extracted twicewith EtOAc. The combined organic layers were dried over MgSO₄, filtered,and evaporated under reduced pressure. The residue was dried undervacuum at 50° C. to give2-((3-(4-(tert-butoxy)-4-oxobutoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)aceticacid 2c (3.22 g) as a thick brown oil.

Synthesis of Intermediate 2d

N,N-Diisopropylethylamine (1.58 mL, 9.57 mmol) was added to a solutionof2-((3-(4-(tert-butoxy)-4-oxobutoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)aceticacid 2c (1.44 g, 3.19) and 4-methyl-6-(trifluoromethyl)indoline 2a (953mg, 3.51 mmol) in dry DMF (30 mL). HATU (1.82 g, 4.78 mmol) was addedand the reaction mixture was stirred at room temperature for 2 h. Thereaction mixture was poured out into water (400 mL) and the whitesuspension was extracted with EtOAc. The aqueous layer was saturated bythe addition of NaCl and extracted again with EtOAc. The combinedorganic layers were washed with brine, water, dried over MgSO₄ andevaporated under reduced pressure. The residue was purified by columnchromatography on silica gel (100 g) using a gradient of EtOAc:EtOH(3:1)/heptane 0/100 to 60/40). The product fractions were combined andevaporated under reduced pressure. The residue (1.41 g) was purified viapreparative HPLC (Stationary phase: RP XBridge® Prep C18 OBD—10 μm,50×150 mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN). Theproduct fractions were combined and evaporated under reduced pressure toprovide tert-butyl4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate2d (808 mg) as a white solid.

Synthesis of Compound 2 and Chiral Separation into Enantiomers 2A and 2B

Tert-butyl4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate2d (808 mg, 1.28 mmol) was mixed with 4M HCl in dioxane (9.6 mL) and themixture was stirred at room temperature for 15 h. Nitrogen gas wasbubbled through the reaction mixture for 30 min. The solvent wasevaporated under reduced pressure to give4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 2, 735 mg) as a light brown solid. The enantiomers ofCompound 2 (735 mg) were separated via preparative chiral SFC(Stationary phase: Chiralcel® Diacel OD 20×250 mm, mobile phase: CO₂,EtOH+0.4% iPrNH₂). The product fractions were combined and evaporatedunder reduced pressure to give Enantiomer 2A as the first eluted productand Enantiomer 2B as the second eluted product. Both residues were mixedwith EtOAc and water. The mixture was acidified to pH 1-2 with 1N HCl.The layers were separated and the aqueous layer was extracted twice withEtOAc. The combined organic layers were washed with water, dried overMgSO₄, filtered, and evaporated under reduced pressure. The residue wasdried under vacuum at 50° C. to give Enantiomer 2A (216 mg) andEnantiomer 2B (184 mg), respectively.

Compound 2:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (br quin, J=6.9 Hz, 2H) 2.25 (s,3H) 2.33 (br t, J=7.1 Hz, 2H) 3.07-3.20 (m, 2H) 3.62 (s, 3H) 3.84 (br t,J=6.4 Hz, 2H) 3.97-4.09 (m, 1H) 4.48-4.60 (m, 1H) 5.57 (br d, J=8.8 Hz,1H) 5.76 (t, J=1.8 Hz, 1H) 5.90-5.99 (m, 2H) 6.43 (br d, J=8.8 Hz, 1H)7.25 (s, 1H) 7.44 (d, J=8.4 Hz, 2H) 7.56 (br d, J=8.4 Hz, 2H) 8.22 (s,1H) 12.15 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.14 min, MH⁺ 577

Enantiomer 2A:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.8 Hz, 2H) 2.25 (s, 3H)2.34 (t, J=7.3 Hz, 2H) 3.05-3.23 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.4 Hz,2H) 4.03 (td, J=10.2, 7.3 Hz, 1H) 4.54 (td, J=10.2, 6.2 Hz, 1H) 5.57 (d,J=8.8 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.91-5.99 (m, 2H) 6.42 (d, J=8.8Hz, 1H) 7.24 (s, 1H) 7.44 (d, J=8.4 Hz, 2H) 7.56 (d, J=8.8 Hz, 2H) 8.22(s, 1H) 12.17 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.26 min, MH⁺ 577

[α]_(D) ²⁰: −39.00 (c 0.438, DMF)

Chiral SFC (method SFC-B): R_(t) 5.11 min, MH⁺ 577 chiral purity 100%.

Enantiomer 2B:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.88 (quin, J=6.9 Hz, 2H) 2.25 (s, 3H)2.34 (t, J=7.3 Hz, 2H) 3.06-3.24 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.4 Hz,2H) 3.97-4.11 (m, 1H) 4.55 (td, J=10.3, 6.8 Hz, 1H) 5.58 (d, J=8.4 Hz,1H) 5.77 (t, J=2.0 Hz, 1H) 5.92-5.99 (m, 2H) 6.43 (d, J=8.8 Hz, 1H) 7.25(s, 1H) 7.41-7.50 (m, 2H) 7.52-7.60 (m, 2H) 8.23 (s, 1H) 12.17 (brs, 1H)

LC/MS (method LC-C): R_(t) 1.25 min, MH⁺ 577

[α]_(D) ²⁰: +47.1° (c 0.384, DMF)

Chiral SFC (method SFC-B): R_(t) 8.00 min, MH⁺ 577 chiral purity 99.6%.

Example 3: Synthesis of4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 3) and Chiral Separation into Enantiomers 3A and 3B

Synthesis of Intermediate 3a

At 0° C., BH₃—Pyridine (10.45 mL, 103.4 mmol) was added slowly to asolution of 5-fluoro-6-(trifluoromethyl)-1H-indole [CAS 1493800-10-4](7.0 g, 34.5 mmol) in EtOH (45 mL). 6N HCl (105 mL) was added dropwisewhile maintaining the temperature below 10° C. The mixture was stirredat 0° C. for 3 h. Water was added and the mixture was basified to pH 8.5with a concentrated solution of NaOH (temperature below 20° C.). EtOAcwas added. The organic layer was separated, washed with water, driedover MgSO₄, filtered and the solvent was evaporated under reducedpressure. Toluene was added and removed under reduced pressure (toeliminate traces of pyridine). The residue was purified by flashchromatography on silica gel (20-45 μm, 120 g, CH₂Cl₂/MeOH 98.5/1.5).The pure fractions were combined and the solvent was concentrated underreduced pressure to give 5-fluoro-6-(trifluoromethyl)indoline 3a (3.5g).

Synthesis of Intermediate 3b

A mixture of 5-fluoro-6-(trifluoromethyl)indoline 3a (500 mg, 2.44mmol), 2-(4-chlorophenyl)acetic acid [CAS 1878-66-6] (457 mg, 2.64mmol), HATU (1.39 g, 3.66 mmol) and diisopropylethylamine (1.2 mL, 7.31mmol) in DMF (10 mL) was stirred at room temperature for 12 h. Themixture was poured out into ice-water, the precipitate was filtered offand taken up with CH₂Cl₂. The organic layer was dried over MgSO₄ andconcentrated under reduced pressure. The compound was crystallized fromCH₃CN and dried to give2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)ethanone3b (854 mg).

Synthesis of Intermediate 3c

At −78° C., under a N₂ flow, LiHMDS 1M in THF (4.78 mL, 4.78 mmol) wasadded dropwise to a mixture of2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)ethanone3b (854 mg, 2.39 mmol) in THF (7 mL). TMSCl (485 μL, 3.82 mmol) wasadded dropwise. The mixture was stirred for 15 min at −78° C. and asolution of N-bromosuccinimide (510 mg, 2.87 mmol) in THF (7 mL) wasadded dropwise. After stirring for 2 h at −78° C., the reaction wasquenched with a saturated aqueous solution of NH₄Cl. EtOAc was added andthe organic layer was separated, dried over MgSO₄, filtered and thesolvent was evaporated under reduced pressure. The residue was purifiedby flash chromatography on silica gel (15-40 μm, 40 g, CH₂Cl₂/heptane50/50). The pure fractions were combined and the solvent wasconcentrated under reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)ethanone3c (820 mg).

Synthesis of Intermediate 3d

A mixture of2-bromo-2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)ethanone3c (820 mg, 1.88 mmol), tert-butyl4-(3-amino-5-methoxyphenoxy)-butanoate 1a (528 mg, 1.88 mmol) anddiisopropylethylamine (388 μL, 2.25 mmol) in CH₃CN (20 mL) was stirredat 70° C. for 4 h. The reaction mixture was concentrated under reducedpressure. The residue was taken up with EtOAc. The organic layer waswashed twice with a 1N solution of HCl, water, dried over MgSO₄,filtered and the solvent was removed under reduced pressure. The residuewas purified by flash chromatography on silica gel (15-40 μm, 40 g,CH₂Cl₂ 100%). The pure fractions were combined and the solvent wasconcentrated under reduced pressure to give tert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)-butanoate 3d (1.07 g).

Synthesis of Compound 3 and Chiral Separation into Enantiomers 3A and 3B

A solution of tert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate3d (1.07 g, 1.68 mmol) in HCl (4M in dioxane) (20 mL) was stirred at 5°C. for 3 h and at room temperature for 12 h. The precipitate wasfiltered off, washed with diisopropyl ether and dried. The residue waspurified via reverse phase chromatography (Stationary phase: YMC-actusTriart-C18 10 μm 30×150 mm, mobile phase: gradient from 65% NH₄HCO₃0.2%, 35% CH₃CN to 25% NH₄HCO₃ 0.2%, 75% CH₃CN) to provide Compound 3(540 mg). An analytical sample (30 mg) was further purified via reversephase chromatography (Stationary phase: YMC-actus Triart-C18 10 μm30×150 mm, mobile phase: gradient from 65% NH₄HCO₃ 0.2 35% CH₃CN to 25%NH₄HCO₃ 0.2%, 75% CH₃CN) to give4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 3, 30 mg, 0.16 H₂O). The remaining amount of Compound 3(510 mg) was used for chiral separation of the enantiomers viaPreparative Chiral SFC (Stationary phase: Whelk 01 S,S 5 μm 250×30 mm,mobile phase: 60% CO₂, 40% MeOH). The first eluted enantiomer (250 mg)was further purified by flash chromatography on silica gel (20-45 μm, 24g, CH₂Cl₂/MeOH 98/2). The pure fractions were combined and the solventwas concentrated under reduced pressure to give, after solidification inheptane/diisopropyl ether, Enantiomer 3A (170 mg). The second elutedenantiomer (249 mg) was solidified in heptane/diisopropyl ether to giveEnantiomer 3B (182 mg).

Compound 3:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.78-1.92 (m, 2H) 2.26 (br s, 2H)3.15-3.31 (m, 2H) 3.61 (s, 3H) 3.84 (br s, 2H) 4.02 (br d, J=7.88 Hz,1H) 4.54 (br d, J=5.99 Hz, 1H) 5.58 (br d, J=8.51 Hz, 1H) 5.76 (br s,1H) 5.90-5.99 (m, 2H) 6.42 (br d, J=8.51 Hz, 1H) 7.44 (br d, J=7.88 Hz,3H) 7.55 (br d, J=7.88 Hz, 2H) 8.38 (br d, J=6.31 Hz, 1H) 11.60-12.92(m, 1H)

LC/MS (method LC-A): R_(t) 2.94 min, MH⁺ 581 Melting point: 206° C.

Enantiomer 3A:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.86 Hz, 2H) 2.29-2.39 (m,2H) 3.18-3.30 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.46 Hz, 2H) 4.03 (td,J=10.25, 7.25 Hz, 1H) 4.54 (td, J=10.17, 6.15 Hz, 1H) 5.58 (d, J=8.51Hz, 1H) 5.76 (s, 1H) 5.95 (br d, J=11.35 Hz, 2H) 6.43 (d, J=8.83 Hz, 1H)7.43-7.48 (m, 3H) 7.55 (d, J=8.51 Hz, 2H) 8.39 (d, J=6.31 Hz, 1H)12.08-12.27 (m, 1H)

LC/MS (method LC-A): R_(t) 2.95 min, MH⁺ 581

[α]_(D) ²⁰: −48.9° (c 0.315, DMF)

Chiral SFC (method SFC-G): R_(t) 1.65 min, MH⁺ 581 chiral purity 100%.

Enantiomer 3B:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.54 Hz, 2H) 2.25-2.46 (m,2H) 3.15-3.31 (m, 2H) 3.62 (s, 3H) 3.85 (br t, J=6.31 Hz, 2H) 3.98-4.07(m, 1H) 4.50-4.59 (m, 1H) 5.58 (br d, J=8.83 Hz, 1H) 5.76 (s, 1H) 5.95(br d, J=12.30 Hz, 2H) 6.43 (br d, J=8.83 Hz, 1H) 7.42-7.48 (m, 3H) 7.56(br d, J=8.20 Hz, 2H) 8.39 (br d, J=6.31 Hz, 1H) 11.40-12.54 (m, 1H)

LC/MS (method LC-A): R_(t) 2.94 min, MH⁺ 581

[α]_(D) ²⁰: +47.8° (c 0.27, DMF)

Chiral SFC (method SFC-G): R_(t) 2.14 min, MH⁺ 581 chiral purity 99.43%.

Example 4: Synthesis of4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 4) and Chiral Separation into Enantiomers 4A and 4B

Synthesis of Intermediate 4a

To a solution of 2-methyl-4-(trifluoromethoxy)aniline [CAS 86256-59-9](10.0 g, 52.3 mmol) in dioxane (20 mL) was added trifluoroaceticanhydride (8 mL, 57.2 mmol). The reaction mixture was stirred at roomtemperature for 1 h. The reaction mixture was concentrated under reducedpressure. The residue was partitioned between EtOAc and 1N HCl. Thephases were separated. The organic phase was washed with a saturatedsolution of NaHCO₃ in water, H₂O and brine, dried over Na₂SO₄, filteredand concentrated under reduced pressure to afford 14.7 g of2,2,2-trifluoro-N-(2-methyl-4-(trifluoromethoxy)phenyl)acetamide 4a as awhite powder. The compound was used in the next step without furtherpurification.

Synthesis of Intermediate 4c

To acetic anhydride (11.4 mL, 61.1 mmol), cooled at 0° C. was addeddropwise 70% nitric acid (3.9 mL).2,2,2-Trifluoro-N-(2-methyl-4-(trifluoromethoxy)phenyl)-acetamide 4a (5g, 17.4 mmol) was added portionwise and the reaction mixture was heatedat 55° C. for 12 h. After cooling to room temperature, the reactionmixture was diluted with EtOAc and washed with H₂O. The organic phasewas washed with brine, dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was dissolved in methanol (46 mL).2M K₂CO₃ (23 mL, 46 mmol) was added and the reaction mixture was heatedat 70° C. for 4 h. More 2M K₂CO₃ (10 mL, 20 mmol) was added and thereaction mixture was heated at 70° C. for 12 h. The reaction mixture waspartially concentrated under reduced pressure to remove methanol. Theresidue was extracted with EtOAc. The organic phase was washed with H₂Oand brine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gelusing a gradient of EtOAc (20% to 50%) in heptane to afford 3.6 g of2-methyl-6-nitro-4-(trifluoromethoxy)aniline 4c as a yellow solid.

Synthesis of Intermediate 4d

To a solution of 2-methyl-6-nitro-4-(trifluoromethoxy)aniline 4c (1.8 g,7.69 mmol) in acetic acid (10.9 mL) was added dropwise a solution ofsodium nitrite (0.806 g, 11.7 mmol) in H₂SO₄/H₂O (2 mL, 1/1). Thereaction mixture was stirred at room temperature for 30 min. H₂O (22 mL)and urea (0.802 g, 13.4 mmol) were added. After 10 min at roomtemperature, a solution of potassium iodide (1.7 g, 10.2 mmol) in H₂O(11 mL) was added dropwise. The reaction mixture was stirred at roomtemperature for 30 min. The yellow solid was filtered off, washed withH₂O and dried to give 2.4 g of2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 4d.

Synthesis of Intermediate 4e

To a solution of 2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 4d(3.5 g, 10.0 mmol) in EtOH (30 mL) was added a solution of NH₄Cl (2.7 g,49.9 mmol) in H₂O (30 mL). The reaction mixture was heated at 50° C.Iron (2.6 g, 46.9 mmol) was added and the reaction mixture was heatedunder reflux for 40 min. After cooling to room temperature, the reactionmixture was filtered through Celite®. The solids were washed with EtOH.The filtrate was partially concentrated under reduced pressure to removeEtOH. The residue was partitioned between EtOAc and a saturated solutionof NaHCO₃ in water. The phases were separated. The organic phase waswashed with H₂O and brine, dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was purified by flash chromatographyon silica gel using a gradient of EtOAc (0% to 25%) in heptane to afford2.9 g of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 4e as a yellow oil.

Synthesis of Intermediate 4f

A solution of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 4e (2.9 g, 9.1mmol) in triethylamine (23 mL) was degassed with argon for 15 min.Dichlorobis(triphenylphosphine)palladium(II) (0.327 g, 0.47 mmol),copper(I) iodide (0.164 g, 0.86 mmol) and trimethylsilylacetylene (1.8mL, 13.1 mmol) were added. The reaction mixture was heated at 65° C. for12 h. After cooling to room temperature, the reaction mixture wasdiluted with H₂O and extracted with EtOAc (3×). The organic phases werecombined, washed with H₂O and brine, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel using a gradient of EtOAc (0% to 20%) inheptane to afford 2.6 g of3-methyl-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 4f asan orange oil.

Synthesis of Intermediate 4g

To a solution of3-methyl-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 4f (2.7g, 9.3 mmol) in NMP (27 mL) was added tBuOK (3.1 g, 27.8 mmol). Thereaction mixture was heated at 80° C. for 4 h. After cooling to roomtemperature, the reaction mixture was diluted with H₂O and extractedwith EtOAc (2×). The organic phases were combined, washed with H₂O andbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gelusing a gradient of EtOAc (0% to 20%) in heptane to afford 1.7 g of4-methyl-6-(trifluoromethoxy)-1H-indole 4g as an orange oil.

Synthesis of Intermediate 4h

At 0° C., BH₃—Pyridine (1.2 mL, 11.6 mmol) was added dropwise to asolution of 4-methyl-6-(trifluoromethoxy)-1H-indole 4g (0.5 g, 2.32mmol) in EtOH (3 mL). 6N HCl (6 mL) was slowly added dropwise whilemaintaining the reaction temperature below 10° C. The mixture wasstirred at 0° C. for 3 h. Water (12 mL) was added and the mixture wasbasified until pH 8-9 with a concentrated solution of NaOH in water (thereaction temperature was kept below 20° C.). The mixture was extractedwith EtOAc. The organic layer was washed with water, dried over MgSO₄,filtered and the solvent was evaporated under reduced pressure. Toluenewas added and the solution was concentrated under reduced pressure togive 450 mg of 4-methyl-6-(trifluoromethoxy)indoline 4h.

Synthesis of Intermediate 4i

N,N-Diisopropylethylamine (1.58 mL, 9.57 mmol) was added to a solutionof2-((3-(4-(tert-butoxy)-4-oxobutoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)aceticacid 2c (1.44 g, 3.19) and 4-methyl-6-(trifluoromethoxy)indoline 4h (846mg, 3.51 mmol) in dry DMF (30 mL). HATU (1.82 g, 4.78 mmol) was addedand the reaction mixture was stirred at room temperature for 2 h. Thereaction mixture was poured out into water (400 mL) and the whitesuspension was extracted with EtOAc. The aqueous layer was saturated bythe addition of NaCl and extracted again with EtOAc. The combinedorganic layers were washed with brine, water, dried over MgSO₄ andevaporated under reduced pressure. The residue was purified by columnchromatography on silica gel (100 g) using a gradient of EtOAc:EtOH(3:1)/heptane 0/100 to 60/40. The product fractions were combined andevaporated under reduced pressure. The residue (1.47 g) was purified viapreparative HPLC (Stationary phase: RP XBridge® Prep C18 OBD—10 μm,50×150 mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN). Theproduct fractions were combined and evaporated under reduced pressure toprovide tert-butyl4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate4i (821 mg) as a white solid.

Synthesis of Compound 4 and Chiral Separation into Enantiomers 4A and 4B

Tert-butyl4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate4i (821 mg, 1.27 mmol) was mixed with 4M HCl in dioxane (9.5 mL) and themixture was stirred at room temperature for 15 h. Nitrogen gas wasbubbled through the reaction mixture for 30 min. The solvent wasevaporated under reduced pressure to give4-(3-((1-(4-chlorophenyl)-2-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 4, 750 mg) as an off-white solid. The enantiomers ofCompound 4 (750 mg) were separated via preparative chiral SFC(Stationary phase: Chiralcel® Diacel OD 20×250 mm, mobile phase: CO₂,EtOH+0.4% iPrNH₂). The product fractions were combined and evaporatedunder reduced pressure to give Enantiomer 4A as the first eluted productand Enantiomer 4B as the second eluted product. Both residues were mixedwith EtOAc and water. The mixture was acidified to pH 1-2 with 1N HCl.The layers were separated and the aqueous layer was extracted twice withEtOAc. The combined organic layers were washed with water, dried overMgSO₄, filtered, and evaporated under reduced pressure. The residue wasdried under vacuum at 50° C. to give Enantiomer 4A (213 mg) andEnantiomer 4B (194 mg) respectively.

Compound 4:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=7.0 Hz, 2H) 2.20 (s, 3H)2.33 (t, J=7.1 Hz, 2H) 2.98-3.16 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.4 Hz,2H) 4.04 (td, J=10.4, 7.0 Hz, 1H) 4.53 (td, J=10.3, 6.4 Hz, 1H) 5.56 (d,J=9.1 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.91-5.98 (m, 2H) 6.45 (d, J=8.8Hz, 1H) 6.87 (s, 1H) 7.38-7.47 (m, 2H) 7.50-7.61 (m, 2H) 7.89 (s, 1H)12.18 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.14 min, MH⁺ 593

Enantiomer 4A:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.9 Hz, 2H) 2.20 (s, 3H)2.34 (t, J=7.3 Hz, 2H) 2.98-3.16 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.4 Hz,2H) 4.05 (td, J=10.4, 7.0 Hz, 1H) 4.53 (td, J=10.3, 6.4 Hz, 1H) 5.56 (d,J=8.8 Hz, 1H) 5.76 (t, J=1.8 Hz, 1H) 5.91-5.99 (m, 2H) 6.45 (d, J=8.8Hz, 1H) 6.88 (s, 1H) 7.38-7.49 (m, 2H) 7.51-7.61 (m, 2H) 7.89 (s, 1H)12.17 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.29 min, MH⁺ 593

[α]_(D) ²⁰: −39.6° (c 0.455, DMF)

Chiral SFC (method SFC-C): R_(t) 3.34 min, MH⁺ 593 chiral purity 100%.

Enantiomer 4B:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.88 (quin, J=6.9 Hz, 2H) 2.20 (s, 3H)2.34 (t, J=7.1 Hz, 2H) 2.98-3.16 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.4 Hz,2H) 4.05 (td, J=10.3, 7.1 Hz, 1H) 4.53 (td, J=10.2, 6.6 Hz, 1H) 5.56 (d,J=8.8 Hz, 1H) 5.76 (t, J=1.8 Hz, 1H) 5.92-5.99 (m, 2H) 6.46 (d, J=8.8Hz, 1H) 6.88 (s, 1H) 7.38-7.49 (m, 2H) 7.50-7.63 (m, 2H) 7.89 (s, 1H)12.16 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.30 min, MH⁺ 593

[α]_(D) ²⁰: +43.7° (c 0.38, DMF)

Chiral SFC (method method SFC-C): R_(t) 3.16 min, MH⁺ 593 chiral purity100%.

Example 5: Synthesis of4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 5) and Chiral Separation into Enantiomers 5A and 5B

Synthesis of Intermediate 5a

A solution of 4-bromo-2-fluoro-1-(trifluoromethoxy)benzene [CAS105529-58-6](98.7 g, 381.1 mmol) in concentrated H₂SO₄ (98%, 200 mL),was cooled to 0° C. with an ice-bath. KNO₃ (43.0 g, 425.3 mmol) wasadded in portions. After addition, the ice-bath was removed and themixture was stirred at room temperature for 16 h. The reaction mixturewas poured out into ice-water (2 L) while stirring. The mixture wasextracted with CH₂Cl₂ (3×500 mL). The combined organic layers werewashed with a saturated aqueous NaHCO₃ solution (2×500 mL), brine (500mL), dried over MgSO₄, filtered and concentrated under reduced pressureto afford 1-bromo-5-fluoro-2-nitro-4-(trifluoromethoxy)benzene 5a (117.2g), which was used in the next step without further purification.

Synthesis of Intermediate 5b

To a stirred suspension of1-bromo-5-fluoro-2-nitro-4-(trifluoromethoxy)benzene 5a (70.0 g, 230mmol) and NH₄Cl (123.2 g, 2.30 mol) in iPrOH (1 L) and water (330 mL)was added reductive iron powder (64.3 g, 1.15 mol) under N₂-atmosphere.The reaction mixture was stirred at 60° C. for 16 h. The reactionmixture was diluted with EtOAc (1 L) and filtered through Celite®. Thefiltrate was concentrated under reduced pressure. The residue waspartitioned between EtOAc (1 L) and water (800 mL). The layers wereseparated and the organic phase was washed with brine (1 L), dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by distillation under reduced pressure (oil pump, b.p. 60-64°C.). 2-Bromo-4-fluoro-5-(trifluoromethoxy)aniline 5b (47.3 g) wasobtained as a yellow oil.

Synthesis of Intermediate 5c

To a mixture of 2-bromo-4-fluoro-5-(trifluoromethoxy)aniline 5b (18.4 g,67.2 mmol) and ethynyl(trimethyl)silane (19.9 g, 202.4 mmol, 28.00 mL)in Et₃N (300 mL) was added CuI (1.28 g, 6.72 mmol) and Pd(PPh₃)₂Cl₂(2.40 g, 3.42 mmol). The reaction mixture was heated under N₂-atmosphereat 90° C. for 16 h. After cooling to room temperature, the mixture wasdiluted with MTBE (300 mL) and filtered through Celite®. The filtratewas concentrated under reduced pressure. The residue was purified byflash chromatography on silica gel (ISCO®, 220 g SepaFlash® Silica FlashColumn, eluent: gradient of 0 to 5% EtOAc in petroleum ether @100mL/min).4-Fluoro-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 5c(16.1 g, 90% purity) was obtained as a brown oil.

Synthesis of Intermediate 5d

A mixture of4-fluoro-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 5c(16.1 g, 55.3 mmol) and tBuOK (18.6 g, 165.8 mmol) in NMP (220.00 mL)was heated at 90° C. for 16 h under N₂-atmosphere. After cooling to roomtemperature, the reaction mixture was poured out into ice-water (1 L)and extracted with MTBE (3×300 mL). The combined organic phases werewashed with water (2×200 mL), brine (300 mL), dried over MgSO₄, filteredand concentrated under reduced pressure. The residue was purified byflash chromatography on silica gel (ISCO®, 120 g SepaFlash® Silica FlashColumn, eluent: gradient of 0 to 5% EtOAc in petroleum ether @ 85mL/min) to afford 5-fluoro-6-(trifluoromethoxy)-1H-indole 5d (11 g)product as a dark-green oil. The residue was combined with anotherfraction (total amount=17.2 g) and further purified by distillationunder reduced pressure (oil pump, b.p. 60-64° C.) to provide5-fluoro-6-(trifluoromethoxy)-1H-indole 5d (14.7 g, 95% purity) as acolorless oil.

Synthesis of Intermediate 5e

At 0° C., BH₃-pyridine (13.8 mL, 136.9 mmol) was added dropwise to asolution of 5-fluoro-6-(trifluoromethoxy)-1H-indole 5d (6 g, 27.4 mmol)in EtOH (40 mL). 6N HCl (90 mL) was added dropwise while maintaining thetemperature below 10° C.

The mixture was stirred at 0° C. for 2 h. Water (100 mL) was added andthe mixture was basified to pH 8-9 with a concentrated solution of NaOHin water (the reaction temperature was kept below 20° C.). The mixturewas extracted with CH₂Cl₂. The organic layer was washed with water,dried over MgSO₄, filtered and the solvent was evaporated under reducedpressure. Toluene was added and the solution was concentrated underreduced pressure to give 5.52 g of 5-fluoro-6-(trifluoromethoxy)indoline5e. The compound was used in the next reaction step without furtherpurification.

Synthesis of Intermediate 5f

To a mixture of 2-bromo-2-(4-chlorophenyl)acetic acid [CAS 3381-73-5](0.61 g, 2.4 mmol), 5-fluoro-6-(trifluoromethoxy)indoline 5e (0.55 g,2.2 mmol) and DMAP (0.027 g, 0.22 mmol) in CH₂Cl₂ (14 mL) was added EDCl(0.51 g, 2.7 mmol). The mixture was stirred at room temperature for 18h. The mixture was diluted with a 10% K₂CO₃ solution in water. Thelayers were decanted. The organic layer was washed with water, driedover MgSO₄, filtered and the solvent was concentrated under reducedpressure to give2-bromo-2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)ethanone5f (1.1 g, purple oil). The compound was used in the next step withoutfurther purification.

Synthesis of Intermediate 5g

A mixture of2-bromo-2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)ethanone5f (1.1 g, 2.2 mmol), tert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate1a (1.0 g, 3.3 mmol) and diisopropylethylamine (1.5 mL, 8.7 mmol) inCH₃CN (29 mL) was stirred at 80° C. for 18 h. The reaction mixture wasconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel (30 μm, 40 g, heptane/EtOAc gradient 85/15to 75/25). The fractions containing the expected compound were combinedand the solvent was concentrated under reduced pressure to givetert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate5g (480 mg, 57% purity by LC/MS).

Synthesis of Compound 5 and Chiral Separation into Enantiomers 5A and 5B

A mixture of tert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate5g (0.48 g, 0.42 mmol, 57% purity) in HCl (4M in dioxane) (4.6 mL) wasstirred at room temperature for 18 h. The mixture was concentrated underreduced pressure, taken up in Et₃N (5 mL) and concentrated again invacuo. The residue was purified by flash chromatography on silica gel(30 μm, 24 g, CH₂Cl₂/MeOH gradient 99/1 to 96/4). The pure fractionswere combined and evaporated to dryness. The residue (150 mg) wasfurther purified via Reverse Phase HPLC (Stationary phase: YMC-actusTriart-C18 10 μm 30×150 mm, mobile phase: gradient from 65% NH₄HCO₃0.2%, 35% CH₃CN to 25% NH₄HCO₃ 0.2%, 75% CH₃CN) to give4-(3-((1-(4-chlorophenyl)-2-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 5, 71 mg). The enantiomers (55 mg) were separated viachiral SFC (Stationary phase: Chiralcel® OD-H 5 μm 250×20 mm, mobilephase: 55% CO₂, 45% MeOH) to give, after freeze-drying from a solventmixture of CH₃CN/water the first eluted Enantiomer 5A (25 mg, whitesolid) and the second eluted Enantiomer 5B (25 mg, white solid).

Compound 5:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.86 (quin, J=6.70 Hz, 2H) 2.24-2.43 (m,2H) 3.06-3.25 (m, 2H) 3.61 (s, 3H) 3.84 (br t, J=6.31 Hz, 2H) 3.94-4.13(m, 1H) 4.46-4.57 (m, 1H) 5.56 (br d, J=8.83 Hz, 1H) 5.75 (s, 1H) 5.93(s, 1H) 5.95 (s, 1H) 6.45 (br d, J=8.83 Hz, 1H) 7.44 (br d, J=8.20 Hz,3H) 7.54 (br d, J=8.20 Hz, 2H) 8.16 (br d, J=6.62 Hz, 1H) 12.12 (br s,1H)

LC/MS (method LC-A): R_(t) 3.00 min, MH⁺ 597

Enantiomer 5A:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.86 (quin, J=6.94 Hz, 2H) 2.25-2.44 (m,2H) 3.06-3.26 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.46 Hz, 2H) 4.05 (td,J=10.32, 7.09 Hz, 1H) 4.48-4.55 (m, 1H) 5.56 (d, J=8.83 Hz, 1H) 5.76 (t,J=1.89 Hz, 1H) 5.94 (br d, J=11.98 Hz, 2H) 6.45 (d, J=8.83 Hz, 1H)7.42-7.46 (m, 3H) 7.54 (d, J=8.20 Hz, 2H) 8.16 (br d, J=6.94 Hz, 1H)12.01 (br s, 1H)

LC/MS (method LC-A): R_(t) 3.00 min, MH⁺ 597

[α]_(D) ²⁰: −35.8° (c 0.257, DMF)

Chiral SFC (method SFC-H): R_(t) 1.34 min, MH⁺ 597 chiral purity 100%.

Enantiomer 5B:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.85 (quin, J=6.86 Hz, 2H) 2.27 (t,J=7.25 Hz, 2H) 3.10-3.31 (m, 2H) 3.61 (s, 3H) 3.78-3.90 (m, 2H) 4.05(td, J=10.40, 7.25 Hz, 1H) 4.52 (td, J=10.32, 6.46 Hz, 1H) 5.57 (d,J=8.83 Hz, 1H) 5.75 (t, J=1.89 Hz, 1H) 5.94 (br d, J=16.39 Hz, 2H) 6.45(d, J=8.83 Hz, 1H) 7.41-7.46 (m, 3H) 7.55 (d, J=8.51 Hz, 2H) 8.16 (br d,J=6.94 Hz, 1H)

LC/MS (method LC-A): R_(t) 3.00 min, MH⁺ 597

[α]_(D) ²⁰: +52.8° (c 0.231, DMF)

Chiral SFC (method SFC-H): R_(t) 3.14 min, MH⁺ 597 chiral purity 100%.

Example 6: Synthesis of4-(3-((1-(4-fluoro-2-methoxyphenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 6) and Chiral Separation into Enantiomers 6A and 6B

Synthesis of Intermediate 6a

A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (2 g, 9.84mmol), 2-(4-fluoro-2-methoxyphenyl)acetic acid [CAS 886498-61-9] (2.17g, 10.8 mmol), HATU (5.62 g, 14.8 mmol) and diisopropylethylamine (4.9mL, 29.5 mmol) in DMF (20 mL) was stirred at room temperature for 3 h.Water and ice were added and the precipitate was filtered off and driedto give2-(4-fluoro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone6a (3.44 g).

Synthesis of Intermediate 6b

At −78° C. under a N₂ flow, LiHMDS (18.7 mL, 18.7 mmol) was addeddropwise to a mixture of2-(4-fluoro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone6a (3.44 g, 9.32 mmol) in THF (45 mL). TMSCl (1.42 mL, 11.2 mmol) wasadded dropwise. The mixture was stirred for 15 min at −78° C. andN-bromosuccinimide (1.83 g, 10.2 mmol) in THF (35 mL) was addeddropwise. After stirring for 2 h at −78° C., the reaction was quenchedwith a saturated NH₄Cl solution. The mixture was extracted with EtOAc,dried over MgSO₄, filtered and the solvent was concentrated underreduced pressure to give2-bromo-2-(4-fluoro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone6b (4.48 g). The crude compound was used without further purification inthe next step.

Synthesis of Intermediate 6c

A mixture of2-bromo-2-(4-fluoro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone6b (2.0 g, 4.46 mmol), tert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate1a (1.26 g, 4.46 mmol) and diisopropylethylamine (1.15 mL, 6.69 mmol) inCH₃CN (45 mL) was stirred at 80° C. for 5 h. The reaction mixture wasconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel (15-40 μm, 40 g, heptane/EtOAc 85/15). Thefractions containing expected compound were combined and the solvent wasconcentrated under reduced pressure to give tert-butyl4-(3-((1-(4-fluoro-2-methoxyphenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate6c (1.6 g, 67% purity by LC/MS).

Synthesis of Compound 6 and Chiral Separation into Enantiomers 6A and 6B

A solution of tert-butyl4-(3-((1-(4-fluoro-2-methoxyphenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate6c (1.5 g, 2.31 mmol) in HCl (4M in dioxane) (15 mL) was stirred at 5°C. for 2 h and at room temperature for 3 h. The solvent was concentratedunder reduced pressure and 3N NaOH were added until neutral pH wasobtained. The solution was extracted with EtOAc. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on silica gel (20-45 μm, 40g, CH₂Cl₂/MeOH gradient 99.5/0.5 to 95/5). The pure fractions werecombined and the solvent was concentrated under reduced pressure toprovide Compound 6 (646 mg). A small fraction was crystallized fromCH₃CN/diisopropyl ether to give4-(3-((1-(4-fluoro-2-methoxyphenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 6, 35 mg). The remaining amount (600 mg) was used forchiral separation of the enantiomers via chiral SFC (Stationary phase:Chiralcel® OD-H 5 μm 250×20 mm, mobile phase: 60% CO₂, 40% MeOH). Toprovide Enantiomer 6A as the first eluted product and Enantiomer 6B asthe second eluted product.

Both enantiomers were further purified by flash chromatography on silicagel (20-45 μm, 12 g, CH₂Cl₂/MeOH gradient 100/0 to 95/5). The purefractions were combined and the solvent was concentrated under reducedpressure to give, after solidification in diisopropyl ether/pentane (+afew drops of CH₃CN), Enantiomer 6A (108 mg) and Enantiomer 6B (108 mg),respectively.

Compound 6:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.82 Hz, 2H) 2.33 (t,J=7.33 Hz, 2H) 3.08-3.27 (m, 2H) 3.61 (s, 3H) 3.78-3.91 (m, 5H)3.92-4.02 (m, 1H) 4.33-4.42 (m, 1H) 5.59 (d, J=8.59 Hz, 1H) 5.75 (s, 1H)5.87 (br d, J=7.07 Hz, 2H) 6.39 (br d, J=8.59 Hz, 1H) 6.78 (td, J=8.46,2.27 Hz, 1H) 6.94-7.02 (m, 2H) 7.29-7.35 (m, 2H) 8.03 (s, 1H) 12.14 (brs, 1H)

LC/MS (method LC-B): R_(t) 2.76 min, MH⁺ 593 Melting point: 164° C.

Enantiomer 6A:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.78 Hz, 2H) 2.31-2.47 (m,2H) 3.10-3.28 (m, 2H) 3.62 (s, 3H) 3.80-3.93 (m, 5H) 3.93-4.06 (m, 1H)4.33-4.44 (m, 1H) 5.59 (br d, J=8.51 Hz, 1H) 5.76 (s, 1H) 5.88 (br d,J=8.83 Hz, 2H) 6.39 (br d, J=8.83 Hz, 1H) 6.79 (td, J=8.43, 2.05 Hz, 1H)6.95-7.04 (m, 2H) 7.30-7.37 (m, 2H) 8.03 (s, 1H) 12.16 (br s, 1H)

LC/MS (method LC-A): R_(t) 2.86 min, MH⁺ 593

[α]_(D) ²⁰: −37.3° (c 0.255, DMF)

Chiral SFC (method SFC-I): R_(t) 1.03 min, MH⁺ 593 chiral purity 100%.

Enantiomer 6B:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.86 Hz, 2H) 2.30-2.45 (m,2H) 3.09-3.26 (m, 2H) 3.62 (s, 3H) 3.80-3.93 (m, 5H) 3.93-4.06 (m, 1H)4.33-4.44 (m, 1H) 5.59 (br d, J=8.51 Hz, 1H) 5.76 (s, 1H) 5.88 (br d,J=8.83 Hz, 2H) 6.39 (br d, J=8.51 Hz, 1H) 6.79 (td, J=8.43, 2.05 Hz, 1H)6.95-7.04 (m, 2H) 7.30-7.37 (m, 2H) 8.03 (br s, 1H), 12.18 (br s, 1H)

LC/MS (method LC-A): R_(t) 2.88 min, MH⁺ 593

[α]_(D) ²⁰: +32.7° (c 0.294, DMF)

Chiral SFC (method SFC-I): R_(t) 1.82 min, MH⁺ 593 chiral purity 99.56%.

Example 7: Synthesis of4-(3-((1-(4-chlorophenyl)-1-deuterio-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 7-D) and Chiral Separation into Enantiomers 7A-D and 7B-D

Synthesis of Intermediate 7a

A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (2 g, 9.84mmol), 2-(4-chlorophenyl)acetic acid [CAS 1878-66-6] (1.85 g, 10.8mmol), HATU (5.6 g, 14.8 mmol) and diisopropylethylamine (4.9 mL, 29.5mmol) in DMF (40 mL) was stirred at room temperature for 12 h. Water wasadded and the precipitate was filtered off. The residue was taken upwith EtOAc. The organic solution was washed with a 10% aqueous solutionof K₂CO₃, brine, dried over MgSO₄, filtered and the solvent wasevaporated under reduced pressure. The residue was purified bychromatography on silica gel (15-40 μm, 80 g, heptane/EtOAc gradient90/10 to 60/40). The pure fractions were combined and the solvent wasconcentrated under reduced pressure to give2-(4-chlorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 7a (3g).

Synthesis of Intermediate 7b

At −78° C., under N₂ flow, LiHMDS 1.5 M in THF (11.2 mL, 16.9 mmol) wasadded dropwise to a mixture of2-(4-chlorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 7a (3 g,8.43 mmol) in THF (50 mL). The mixture was stirred for 15 min at −78° C.and a solution of N-bromosuccinimide (1.65 g, 9.3 mmol) in THF (30 mL)was added dropwise. After stirring for 2 h at −78° C., the reaction wasquenched with a saturated solution of NH₄Cl. The mixture was extractedwith EtOAc. The organic layer was separated, dried over MgSO₄, filteredand the solvent was evaporated under reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone7b (3.6 g). The compound was used as such in the next step.

Synthesis of Intermediate 7c

A mixture of2-bromo-2-(4-chlorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone7b (3.6 g, 8.3 mmol), tert-butyl 4-(3-amino-5-methoxyphenoxy)-butanoate1a (2.3 g, 8.3 mmol) and diisopropylethylamine (1.7 mL, 9.94 mmol) inCH₃CN (80 mL) was stirred at 70° C. for 4 h. The mixture wasconcentrated under reduced pressure, diluted with EtOAc, and washed with1N HCl and water. The organic phase was separated, dried over MgSO₄,filtered and the solvent was evaporated under reduced pressure. Thecompound was purified by flash chromatography on silica gel (15-40 μm,120 g, heptane/EtOAc 80/20). The pure fractions were combined andevaporated to dryness to give, after crystallization from diisopropylether, tert-butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(trifluoro-methoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate7c (2.6 g).

Synthesis of Compound 7 and Chiral Separation into Enantiomers 7A and 7B

A solution of tert-butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(trifluoromethoxy)-indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate7c (2.4 g, 3.8 mmol) in HCl (4M in dioxane) (24 mL) was stirred at 5° C.for 3 h and at room temperature for 3 h. The precipitate was filteredoff and dried to afford4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid as an HCl salt (Compound 7, 2 g, 0.8 equiv. HCl, 0.07 equiv. H₂O).Compound 7 (2 g, HCl salt) was neutralized prior to chiral separation bytreatment of a solution of Compound 7 (HCl salt) with 1N NaOH andevaporation of the organic layer under reduced pressure. The enantiomerswere separated via Preparative Chiral SFC (Stationary phase: Chiralcel®OD-H 5 μm 250×30 mm, mobile phase: 50% CO₂, 50% iPrOH (+0.3% iPrNH₂))and further purified via Preparative achiral SFC (Stationary phase:Cyano® 6 μm 150×21.2 mm, mobile phase: 80% CO₂, 20% MeOH (+0.3%iPrNH₂)). The product fractions were combined and evaporated underreduced pressure. The two enantiomers were taken up with EtOAc andwashed with 1N HCl. The organic layers were separated, dried over MgSO₄,filtered and the solvent was evaporated under reduced pressure. Thefirst eluted enantiomer was solidified from ether/diisopropyl ether togive Enantiomer 7A (616 mg). The second eluted enantiomer was solidifiedfrom ether/diisopropyl ether to give Enantiomer 7B (715 mg).

Compound 7:

¹H NMR (500 MHz, DMSO-d6) δ ppm 1.87 (quin, J=6.9 Hz, 2H) 2.34 (t, J=7.3Hz, 2H) 3.07-3.28 (m, 2H) 3.62 (s, 3H) 3.85 (t, J=6.5 Hz, 2H) 4.04 (td,J=10.5, 7.1 Hz, 1H) 4.52 (td, J=10.3, 6.5 Hz, 1H) 5.57 (s, 1H) 5.76 (t,J=2.2 Hz, 1H) 5.90-6.00 (m, 2H) 7.01 (dd, J=8.2, 1.6 Hz, 1H) 7.33 (d,J=8.2 Hz, 1H) 7.41-7.48 (m, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.03 (s, 1H)

LC/MS (method LC-B): R_(t) 2.70 min, MH⁺ 579 Melting point: 150° C.

Enantiomer 7A:

¹H NMR (500 MHz, DMSO-d6) δ ppm 1.87 (quin, J=6.7 Hz, 2H) 2.34 (br t,J=7.3 Hz, 2H) 3.08-3.27 (m, 2H) 3.62 (s, 3H) 3.85 (br t, J=6.3 Hz, 2H)3.99-4.11 (m, 1H) 4.47-4.57 (m, 1H) 5.57 (br s, 1H) 5.76 (s, 1H) 5.95(br d, J=10.1 Hz, 2H) 6.45 (br s, 1H) 7.01 (br d, J=7.6 Hz, 1H) 7.34 (brd, J=7.9 Hz, 1H) 7.44 (br d, J=8.5 Hz, 2H) 7.55 (br d, J=8.2 Hz, 2H)8.04 (br s, 1H) 12.12 (br s, 1H)

LC/MS (method LC-A): R_(t) 2.95 min, MH⁺ 579

[α]_(D) ²⁰: −48.5° (c 0.27, DMF)

Chiral SFC (method SFC-D): R_(t) 1.13 min, MH⁺ 579, chiral purity 100%.

Enantiomer 7B:

¹H NMR (500 MHz, DMSO-d6) δ ppm 1.87 (br t, J=6.8 Hz, 2H) 2.34 (br t,J=7.3 Hz, 2H) 3.09-3.27 (m, 2H) 3.62 (s, 3H) 3.85 (br t, J=6.1 Hz, 2H)3.99-4.10 (m, 1H) 4.46-4.59 (m, 1H) 5.57 (s, 1H) 5.76 (br s, 1H) 5.95(br d, J=10.1 Hz, 2H) 6.45 (br s, 1H) 7.01 (br d, J=7.9 Hz, 1H) 7.34 (brd, J=7.9 Hz, 1H) 7.44 (br d, J=8.2 Hz, 2H) 7.55 (br d, J=8.2 Hz, 2H)8.04 (br s, 1H) 12.12 (br s, 1H)

LC/MS (method LC-A): R_(t) 2.94 min, MH⁺ 579

[α]_(D) ²⁰: +42.9° (c 0.28, DMF)

Chiral SFC (method SFC-D): R_(t) 2.13 min, MH⁺ 579, chiral purity 100%.

Synthesis of Deuterated Compound 7-D and Chiral Separation intoEnantiomers 7A-D and 7B-D

Copper(II) acetate (241 mg, 1.33 mmol) was added in one portion to asolution of Enantiomer 7A (384 mg, 0.663 mmol) in CH₃CN (15 mL) at roomtemperature. The reaction mixture was heated in a sealed tube undermicrowave irradiation at 130° C. for 2 h. The reaction mixture wasevaporated to dryness under reduced pressure and the residue was takenup with CH₂Cl₂ and water. The layers were separated. The aqueous layerwas extracted again with CH₂Cl₂. The combined organic layers were washedwith brine and water, dried over MgSO₄, filtered, and evaporated underreduced pressure. The residue, containing crude intermediate 7d wasdissolved in MeOH (20 mL). Sodium cyanoborodeuteride (349 mg, 5.31 mmol)and two drops of acetic acid were added and the reaction mixture wasstirred at room temperature for 55 h. Additional sodiumcyanoborodeuteride (48 mg, 0.663 mmol) and a few drops of acetic acidwere added and the reaction mixture was stirred for 7 h at roomtemperature. The solvent was evaporated under reduced pressure. Theresidue was mixed with water and Et₂O. The biphasic system was acidifiedto pH 1-2 by the addition of 1N HCl. The layers were separated. Theaqueous layer was extracted again with Et₂O. The combined organic layerswere dried over MgSO₄ and the solvent was evaporated under reducedpressure. The residue was dried under vacuum at 50° C. to give racemic4-(3-((1-(4-chlorophenyl)-1-deuterio-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 7-D, 242 mg) as a white solid.

The enantiomers of Compound 7-D (242 mg) were separated via preparativeSFC (Stationary phase: Kromasil (R,R) Whelk-01 10/100, mobile phase:CO₂, EtOH+0.4% iPrNH₂). The product fractions were combined andevaporated under reduced pressure to provide Enantiomer 7A-D as thefirst eluted product and Enantiomer 7B-D as the second eluted product.Both enantiomers were mixed with in Et₂O and water. The mixture wasacidified to pH 1-2 with 1N HCl. The layers were separated and theaqueous layer was extracted twice with Et₂O. The combined organic layerswere washed with water, dried over MgSO₄, filtered, evaporated underreduced pressure and dried under vacuum at 50° C. to give Enantiomer7A-D (85 mg, 92% deuterated according to ¹H HMR) and Enantiomer 7B-D (77mg, 92% deuterated according to ¹H HMR) as off-white solids.

Enantiomer 7A-D:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=7.0 Hz, 2H) 2.34 (t, J=7.1Hz, 2H) 3.07-3.25 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.4 Hz, 2H) 4.05 (td,J=10.3, 7.1 Hz, 1H) 4.52 (td, J=10.3, 6.4 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H)5.92-5.98 (m, 2H) 6.45 (s, 1H) 7.01 (dd, J=8.1, 1.5 Hz, 1H) 7.33 (d,J=8.1 Hz, 1H) 7.39-7.49 (m, 2H) 7.51-7.60 (m, 2H) 8.03 (s, 1H) 12.17 (brs, 1H)

LC/MS (method LC-C): R_(t) 1.13 min, MH⁺ 580

[α]_(D) ²⁰: +54.2° (c 0.41, DMF)

Chiral SFC (method SFC-E): R_(t) 5.51 min, MH⁺ 580, chiral purity 100%.

Enantiomer 7B-D:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=6.9 Hz, 2H) 2.34 (t, J=7.3Hz, 2H) 3.07-3.25 (m, 2H) 3.61 (s, 3H) 3.84 (t, J=6.6 Hz, 2H) 4.05 (td,J=10.4, 7.3 Hz, 1H) 4.52 (td, J=10.3, 6.4 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H)5.92-5.98 (m, 2H) 6.45 (s, 1H) 7.01 (dd, J=8.1, 1.5 Hz, 1H) 7.33 (d,J=8.1 Hz, 1H) 7.40-7.49 (m, 2H) 7.51-7.62 (m, 2H) 8.03 (s, 1H) 12.16 (brs, 1H)

LC/MS (method LC-C): R_(t) 1.10 min, MH⁺ 580

[α]_(D) ²⁰: −50.1 (c 0.459, DMF)

Chiral SFC (method SFC-E): R_(t) 6.10 min, MH⁺ 580, chiral purity 100%.

Example 8: Synthesis of4-(3-((1-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 8)

Synthesis of Intermediate 8a

To a mixture of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate [CAS1261826-30-5](5.2 g, 24.2 mmol) and cesium carbonate (15.8 g, 48.5 mmol)in DMF (90 mL) at 10° C. was added(2-bromoethoxy)(tert-butyl)dimethylsilane [CAS 86864-60-0](6.26 mL, 29.1mmol). The reaction mixture was stirred at room temperature overnight.Water was added and the mixture was extracted with EtOAc. The organicphase was dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gel(15-40 μm, 80 g, heptane/EtOAc 80/20). The pure fractions were combinedand the solvent was removed under reduced pressure to give ethyl2-(2-(2-((tert-butyl-dimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 8a(7.8 g).

Synthesis of Intermediate 8b

To a cooled (−70° C.) solution of 1M lithium bis(trimethylsilyl)amide inTHF (41.8 mL, 41.8 mmol) was added a solution of ethyl2-(2-(2-((tert-butyldimethylsilyl)oxy)-ethoxy)-4-chlorophenyl)acetate 8a(7.8 g, 20.9 mmol) in THF (45 mL). After stirring for 1 h at −70° C.,chlorotrimethylsilane (4.24 mL, 33.5 mmol) was added. The reactionmixture was stirred at −70° C. for 15 min. N-Bromosuccinimide (4.46 g,25.1 mmol) in THF (45 mL) was added and stirring was continued at −55°C. for 2 h. The reaction mixture was poured out into H₂O and extractedtwice with EtOAc. The organic layers were combined, dried over MgSO₄,filtered and concentrated under reduced pressure to give ethyl2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)-ethoxy)-4-chlorophenyl)acetate8b (10.1 g), which was used in the next step without furtherpurification.

Synthesis of Intermediate 8c

A mixture of ethyl2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate8b (2.0 g, 4.429 mmol), tert-butyl 4-(3-amino-5-methoxyphenoxy)butanoate1a (1.62 g, 5.76 mmol) and diisopropylethylamine (1.53 mL, 8.86 mmol) inCH₃CN (40 mL) was stirred at 50° C. for 12 h. The reaction mixture wasconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel (15-40 μm, 80 g, heptane/EtOAc gradient85/15 to 60/40). The pure fractions were combined and the solvent wasconcentrated under reduced pressure to give tert-butyl4-(3-((1-(2-(2-((tert-butyldimethylsilyl)-oxy)ethoxy)-4-chlorophenyl)-2-ethoxy-2-oxoethyl)amino)-5-methoxyphenoxy)-butanoate8c (1.1 g).

Synthesis of Intermediate 8d

Lithium hydroxide monohydrate (142 mg, 3.37 mmol) in water (7.5 mL) wasadded dropwise to a solution of tert-butyl4-(3-((1-(2-(2-((tert-butyldimethylsilyl)oxy)-ethoxy)-4-chlorophenyl)-2-ethoxy-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate8c (1.1 g, 1.69 mmol) in THF/CH₃OH (1/1) (15 mL) at 10° C. The reactionwas stirred at room temperature for 5 h, diluted with water and cooledto 0° C. The solution was slowly acidified to pH 6-7 with 0.5N HCl, andextracted with EtOAc. The organic layer was dried over MgSO₄, filteredand the solvent was concentrated under reduced pressure to give2-((3-(4-(tert-butoxy)-4-oxobutoxy)-5-methoxyphenyl)amino)-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)aceticacid 8d (675 mg). The compound was used without further purification inthe next step.

Synthesis of Intermediate 8e

To a solution of2-((3-(4-(tert-butoxy)-4-oxobutoxy)-5-methoxyphenyl)amino)-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)aceticacid 8d (675 mg, 1.08 mmol) in DMF (6 mL) were added HATU (617 mg, 1.62mmol), diisopropylethylamine (536 μL, 3.24 mmol) and6-(trifluoromethoxy)indoline [CAS 959235-95-1] (220 mg, 1.08 mmol). Thereaction mixture was stirred at room temperature for 7 days. Thereaction mixture was diluted with water. The precipitate was filteredoff, washed with water and taken up with EtOAc. The organic layer waswashed with a 10% solution of K₂CO₃ and water, dried over MgSO₄,filtered and the solvent was concentrated under reduced pressure to givetert-butyl4-(3-((1-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate8e (385 mg). The compound was used without further purification in thenext reaction step.

Synthesis of Intermediate 8f

Under N₂ flow at 5° C., HCl (4M in dioxane) (1.19 mL, 4.76 mmol) wasadded dropwise to a solution of tert-butyl4-(3-((1-(2-(2-((tert-butyldimethylsilyl)oxy)-ethoxy)-4-chlorophenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate8e (385 mg, 0.476 mmol) in MeOH (5 mL). The reaction was stirred at roomtemperature for 1 h. The mixture was cooled to 0° C., basified with a10% aqueous solution of K₂CO₃ and extracted with EtOAc. The organicphase was separated, dried over MgSO₄, filtered and the solvent wasconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel (15-40 μm, 24 g, CH₂Cl₂/MeOH 99/1). Thepure fractions were combined and the solvent was removed under reducedpressure to give methyl4-(3-((1-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate8f (99 mg).

Synthesis of Compound 8

Lithium hydroxide monohydrate (32 mg, 0.76 mmol) in water (2.5 mL) wasadded dropwise to a solution of methyl4-(3-((1-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate8f (99 mg, 0.152 mmol) in THF (2.5 mL) at 10° C. The reaction wasstirred at room temperature for 18 h, and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gel(20-45 μm, 12 g, CH₂Cl₂/MeOH gradient 99/1 to 90/10). The fractionscontaining expected compound were combined and the solvent was removedunder reduced pressure. A second purification was performed via Reversephase HPLC (Stationary phase: YMC-actus Triart-C18 10 μm 30×150 mm,mobile phase: gradient from 65% NH₄HCO₃ 0.2%, 35% CH₃CN to 25% NH₄HCO₃0.2%, 75% CH₃CN) to give, after freeze drying from a mixture ofwater/CH₃CN (8/2),4-(3-((1-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoic acid (Compound 8, 16 mg).

Compound 8:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.86 (quin, J=6.86 Hz, 2H) 2.28-2.47 (m,2H) 3.10-3.27 (m, 2H) 3.61 (s, 3H) 3.68-3.88 (m, 4H) 4.06-4.23 (m, 3H)4.39 (td, J=10.09, 6.62 Hz, 1H) 5.70-5.76 (m, 2H) 5.91 (br d, J=9.14 Hz,2H) 6.44 (d, J=8.83 Hz, 1H) 6.99-7.03 (m, 2H) 7.12 (d, J=1.89 Hz, 1H)7.34 (d, J=8.20 Hz, 2H) 8.02 (s, 1H)

LC/MS (method LC-B): R_(t) 2.65 min, MH⁺ 639

Example 9: Synthesis of4-(3-((1-(4-chlorophenyl)-2-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 9)

Synthesis of Intermediate 9a

A suspension of 2-chloro-6-methyl-3-(trifluoromethyl)pyridine [CAS1099597-74-6](4.8 g, 24.6 mmol in sodium methoxide (25% in MeOH) (24 mL,105 mmol) was stirred at room temperature for 60 h. The mixture waspoured out into ice-water and extracted twice with Et₂O. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to give 2-methoxy-6-methyl-3-(trifluoromethyl)pyridine9a (4.69 g). The product was used as such in the next step.

Synthesis of Intermediate 9b

HNO₃ (2.32 mL, 49.1 mmol) was added dropwise to a cooled (0° C.)solution of 2-methoxy-6-methyl-3-(trifluoromethyl)pyridine 9a (4.69 g,24.5 mmol) in H₂SO₄ (63.3 mL, 1.128 mol). The reaction mixture wasstirred at 50° C. for 60 h. the reaction mixture was poured outcarefully into ice-water and the mixture was stirred at 0° C. for 30min. The solid was filtered off and washed with water to give2-methoxy-6-methyl-5-nitro-3-(trifluoromethyl)pyridine 9b (4.38 g) as awhite solid.

Synthesis of Intermediate 9c

2-methoxy-6-methyl-5-nitro-3-(trifluoromethyl)pyridine 9b (4.38 g, 18.5mmol) was dissolved in dry DMF (84 mL) under N₂ atmosphere. DMF-DMA(12.2 mL, 91.5 mmol) was added and the reaction mixture was heated at120° C. for 4 h. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the solid residue was purifiedby column chromatography on silica gel (120 g) using a gradient ofpetroleum ether/EtOAc from 100/0 to 60/40). The pure fractions werecombined and the solvent was removed under reduced pressure to give(E)-2-(6-methoxy-3-nitro-5-(trifluoromethyl)pyridin-2-yl)-N,N-dimethylethenamine 9c (4.5 g) as a red solid.

Synthesis of Intermediate 9d

(E)-2-(6-methoxy-3-nitro-5-(trifluoromethyl)pyridin-2-yl)-N,N-dimethylethenamine9c (4.5 g, 15.5 mmol) was dissolved in toluene (87 mL) under N₂atmosphere. Silica gel (4.64 g), iron powder (8.63 g, 154.5 mmol) andacetic acid (35.4 mL) were added and the reaction mixture was stirred at90° C. for 2 h. The reaction mixture was filtered over Celite® and thesolid was rinsed several times with EtOAc. The combined filtrates wereevaporated and the residue was purified by column chromatography onsilica gel (petroleum ether/EtOAc gradient 100/0 to 65/35) to give5-methoxy-6-(trifluoromethyl)-1H-pyrrolo[3,2-b]pyridine 9d (3.1 g) as ayellow solid.

Synthesis of Intermediate 9e

5-methoxy-6-(trifluoromethyl)-1H-pyrrolo[3,2-b]pyridine 9d (2.04 g, 9.44mmol) was dissolved in dry CH₂Cl₂ (90 mL) under N₂ atmosphere. DMAP (123mg, 1.01 mmol) and Boc₂O (2.49 g, 11.4 mmol) were added. The reactionmixture was stirred for 30 min at room temperature, concentrated underreduced pressure and the residue was purified by flash columnchromatography on silica gel (petroleum ether/EtOAc gradient 100/0 to96/4) to give tert-butyl5-methoxy-6-(trifluoromethyl)-1H-pyrrolo[3,2-b]pyridine-1-carboxylate 9e(2.95 g) as a white solid.

Synthesis of Intermediate 9f

tert-butyl5-methoxy-6-(trifluoromethyl)-1H-pyrrolo[3,2-b]pyridine-1-carboxylate 9e(1.45 g, 4.59 mmol) was dissolved in EtOH (30 mL) and the reaction waspurged with nitrogen. Pd/C (10%) (976 mg, 0.917 mmol) was added to thereaction mixture was hydrogenated overnight at 50° C. The reactionmixture was cooled down to room temperature and filtered over Celite®.The filter cake was washed with EtOH and the filtrate was concentratedunder reduced pressure. The residue was purified by flash columnchromatography (petroleum ether/EtOAc gradient 100/0 to 95/5) to givetert-butyl5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridine-1-carboxylate9f (1.2 g) as a white solid.

Synthesis of Intermediate 9g

A solution of tert-butyl5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridine-1-carboxylate9f (1.2 g, 3.77 mmol) in TFA/CH₂Cl₂ (1/1) (19 mL) was stirred at roomtemperature for 1 h. The reaction mixture was diluted with CH₂Cl₂ (60mL), washed with a saturated aqueous Na₂CO₃ solution (60 mL) and brine(60 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography on silica gel (40 g, petroleum ether/EtOAcgradient 80/20 to 40/60) to give5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridine 9g(745 mg) as a yellow solid.

Synthesis of Intermediate 9h

5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridine 9g(350 mg, 1.60 mmol) was dissolved in dry CH₂Cl₂ (6.5 mL) under N₂atmosphere. DMAP (28 mg, 0.229 mmol) and2-bromo-2-(4-chlorophenyl)acetic acid [CAS 3381-73-5](460 mg, 1.84 mmol)were added. EDCl (383 mg, 1.998 mmol) was added and the reaction mixturewas stirred at room temperature for 1 h. The reaction mixture wasdiluted with CH₂Cl₂, cooled to 0° C. and a saturated aqueous solution ofK₂CO₃ was added. The layers were separated and the organic layer waswashed with brine, dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by flash column chromatography onsilica gel (40 g, petroleum ether/EtOAc gradient 100/0 to 60/40). Asecond purification was performed on silica gel (40 g, toluene/Et₂Ogradient 100/0 to 90/10). A third purification was performed (12 g,toluene/Et₂O gradient 98/2 to 97/3). The pure fractions were combinedand concentrated under reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)ethanone9h (407 mg) as pale green foam.

Synthesis of Intermediate 9i

2-bromo-2-(4-chlorophenyl)-1-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)ethanone9h (400 mg, 0.89 mmol) and tert-butyl4-(3-amino-5-methoxyphenoxy)butanoate 1a (300 mg, 1.07 mmol) weredissolved in dry CH₃CN (40 mL) under N₂ atmosphere.Diisopropylethylamine (232 μL, 1.33 mmol) was added and the reactionmixture was heated to 70° C. for 36 h. The reaction mixture was dilutedwith 20 mL of EtOAc, and washed with 1M HCl and brine. The organic layerwas dried over Na₂SO₄, filtered and concentrated under reduced pressure.The residue was purified by flash column chromatography on silica gel(40 g, toluene/EtOAc gradient 100/0 to 94/6). A second purification wasperformed by column chromatography on silica gel (2×12 g, petroleumether/acetone gradient 100/0 to 95/5). The pure fractions were combinedand concentrated under reduced pressure to give tert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate9i (341 mg) as white foam.

Synthesis of Compound 9

Tert-butyl4-(3-((1-(4-chlorophenyl)-2-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoate9i (341 mg, 0.525 mmol) was dissolved under N₂ atmosphere in HCl (4M indioxane) (6.62 mL). The reaction was stirred at room temperature for 3h. The mixture was concentrated under reduced pressure. The residue waspurified by flash column chromatography on silica gel (40 g,toluene/EtOAc/AcOH gradient 99/0/1 to 50/49/1). A second purificationwas performed on silica gel (2×12 g, CH₂Cl₂/MeOH/AcOH gradient 99/0/1 to96/3/1). A third purification was performed on silica gel (12 g,CH₂Cl₂/MeOH/AcOH gradient 98/1/1 to 96.5/2.5/1). The pure fractions werecombined and concentrated under reduced pressure to give4-(3-((1-(4-chlorophenyl)-2-(5-methoxy-6-(trifluoromethyl)-2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-1-yl)-2-oxoethyl)amino)-5-methoxyphenoxy)butanoicacid (compound 9, 72 mg) as white solid.

Compound 9:

¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.84-1.91 (m, 2H) 2.30-2.37 (m, 2H)3.21-3.30 (m, 2H) 3.62 (s, 3H) 3.80-3.89 (m, 2H) 3.94 (s, 3H) 3.98-4.12(m, 1H) 4.56 (td, J=10.64, 6.15 Hz, 1H) 5.58 (d, J=8.83 Hz, 1H) 5.76 (t,J=1.89 Hz, 1H) 5.95 (br d, J=10.72 Hz, 2H) 6.40 (d, J=8.83 Hz, 1H) 7.44(d, J=8.51 Hz, 2H) 7.56 (d, J=8.51 Hz, 2H) 8.53 (s, 1H) 12.06-12.26 (m,1H)

LC/MS (method LC-A): R_(t) 2.87 min, MH⁺ 594

Example 10: Synthesis of4-(3-((1-(4-chlorophenyl)-2-oxo-2-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 10) and Chiral Separation into Enantiomers 10A and 10B

Synthesis of Intermediate 10a

A solution of ethyl 2-(3-amino-5-(trifluoromethyl)thiophen-2-yl)acetate([CAS 860398-39-6] (1.49 g, 5.88 mmol) in CH₃CN (40 mL) was stirred atroom temperature under N₂-atmosphere. NaHCO₃ (0.544 g, 6.47 mmol) and2-(4-chlorophenyl)acetyl chloride ([CAS 25026-34-0] (861 μL, 5.88 mmol)were added, and the reaction mixture was stirred at room temperature for100 min. The mixture was poured out into stirring H₂O (200 mL) andextracted with Et₂O (2×100 mL).

The combined organic layers were washed with brine, dried over MgSO₄,filtered and evaporated under reduced pressure. The residue was purifiedby flash chromatography on silica gel (50 g) using a gradient ofheptane/EtOAc 100/0 to 80/20. The desired fractions were combined,evaporated under reduced pressure and co-evaporated with toluene toprovide ethyl2-(3-(2-(4-chlorophenyl)acetamido)-5-(trifluoromethyl)thiophen-2-yl)acetate10a (1.15 g).

Synthesis of Intermediate 10b

A solution of LiBH₄ 2M in THF (2.59 mL, 5.18 mmol) was added slowly to astirring solution of ethyl2-(3-(2-(4-chlorophenyl)acetamido)-5-(trifluoromethyl)thiophen-2-yl)acetate10a (1.05 g, 2.59 mmol) in 2-Me-THF (20 mL). The reaction mixture wasstirred at room temperature for 18 h. The mixture was poured out into astirring mixture of H₂O (100 mL) and Et₂O (100 mL). 1N HCl (10 mL) wasadded dropwise (foaming), and after stirring for 15 minutes, the layerswere separated. The organic layer was washed with brine, dried overMgSO₄, filtered, and evaporated under reduced pressure. The residue waspurified by flash chromatography on silica gel (25 g) using a gradientof heptane/iPrOH 100/0 to 50/50. The desired fractions were combined,evaporated under reduced pressure, and co-evaporated with toluene. Theresidue was stirred up in toluene (6 mL) at 45° C. for 15 minutes,filtered off at room temperature, washed with toluene (3×), and driedunder vacuum at 50° C. to provide2-(4-chlorophenyl)-N-(2-(2-hydroxyethyl)-5-(trifluoromethyl)thiophen-3-yl)acetamide10b (1.15 g).

Synthesis of Intermediate 10c

Triphenylphosphine (1.02 g, 3.85 mmol) was added to a stirring solutionof2-(4-chlorophenyl)-N-(2-(2-hydroxyethyl)-5-(trifluoromethyl)thiophen-3-yl)acetamide10b (1.0 g, 2.75 mmol) in THF (20 mL) under N₂-atmosphere. Di-tert-butylazodicarboxylate (0.71 g, 3.02 mmol) was added and the resultingsolution was stirred at room temperature for 20 h. The volatiles wereevaporated under reduced pressure and the residue was purified by flashchromatography on silica gel (25 g) using a gradient of CH₂Cl₂/heptane0/100 to 100/0. The desired fractions were combined and concentratedunder reduced pressure to a residual volume of 15 mL. The product wasallowed to crystallize over a period of 4 days. The solids were filteredoff, washed with heptane (4×) and dried under vacuum at 50° C. toprovide2-(4-chlorophenyl)-1-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethanone10c (0.75 g).

Synthesis of Intermediate 10d

At −75° C., under a N₂ flow, LiHMDS 1M in THF (4.34 mL, 4.34 mmol) wasadded dropwise to a mixture of2-(4-chlorophenyl)-1-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethanone10c (750 mg, 2.17 mmol) in 2-Me-THF (30 mL) and the mixture was kept at−75° C. for 20 min. TMSCl (444 μL, 3.47 mmol) was added dropwise. Themixture was stirred for 20 min at −75° C. and a solution ofN-bromosuccinimide (502 mg, 2.82 mmol) in THF (5 mL) was added dropwise.After stirring for 20 min at −75° C., the reaction was quenched with asaturated aqueous solution of NH₄Cl (25 mL). The cooling bath wasremoved and the reaction mixture was stirred until the reactiontemperature reached −15° C. Water (25 mL) and DIPE (25 mL) were addedand the mixture was stirred for 10 min. The organic layer was separatedand the aqueous phase was extracted with Et₂O. The combined organiclayers were dried over MgSO₄, filtered and the solvent was evaporatedunder reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethanone10d (921 mg), which was used as such in the next step.

Synthesis of Intermediate 10e

A mixture of2-bromo-2-(4-chlorophenyl)-1-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethanone10d (921 mg, 2.17 mmol), tert-butyl4-(3-amino-5-methoxyphenoxy)butanoate 1a (1.22 g, 4.34 mmol) anddiisopropylethylamine (747 μL, 4.34 mmol) in 2-butanol (15 mL) wasstirred at 45° C. for 2 h. The reaction mixture was allowed to reachroom temperature, and poured out into stirring water (50 mL). Theproduct was extracted (2×) with Et₂O. The combined organic layers weredried over MgSO₄, filtered, and the solvent was evaporated under reducedpressure and co-evaporated with dioxane (2×). The residue was purifiedby flash chromatography on silica gel (40 g) using a gradient ofheptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions werecombined, evaporated under reduced pressure, and co-evaporated withdioxane (2×) to provide tert-butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethyl)amino)-5-methoxyphenoxy)butanoate10e (1.36 g), which was used as such in the next step.

Synthesis of Compound 10 and Chiral Separation into Enantiomers 10A and10B

tert-Butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethyl)amino)-5-methoxyphenoxy)butanoate10e (1.36 g, 2.17 mmol), was mixed with 4M HCl in dioxane (15 mL) andthe mixture was stirred at room temperature for 20 h. The solids werefiltered off, washed with dioxane (3×), and dried under vacuum at 50° C.The residue (1.4 g) was purified via preparative HPLC (Stationary phase:RP XBridge® Prep C18 OBD—10 μm, 50×150 mm, mobile phase: 0.25% NH₄HCO₃solution in water, CH₃CN). The desired fractions were combined and theorganic volatiles were evaporated under reduced pressure. The remainingaqueous solution was extracted (2×) with a solvent mixture ofEt₂O/2-Me-THF (2/1). The combined organic layers were washed with brine,dried over MgSO₄, filtered, and evaporated under reduced pressure toprovide crude4-(3-((1-(4-chlorophenyl)-2-oxo-2-(2-(trifluoromethyl)-5,6-dihydro-4H-thieno[3,2-b]pyrrol-4-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 10, 0.54 g). An analytical sample (40 mg) was dissolvedin stirring Et₂O (1 mL) and 4M HCl in dioxane (250 μL) was added. Afterstirring for 2 min, the product was filtered off, washed (3×) withEt₂O/dioxane (4/1), and dried under vacuum at 50° C. to provide Compound10 (20 mg).

The enantiomers of Compound 10 (500 mg) were separated via preparativechiral SFC (Stationary phase: Chiralpak® Diacel IC 20×250 mm, mobilephase: CO₂, EtOH). The product fractions of the first eluted enantiomerwere combined, evaporated under reduced pressure and purified by flashchromatography on silica gel (12 g) using a gradient ofheptane/EtOAc:EtOH:AcOH 100/0:0:0 to 60/30:9.8:0.2. The desiredfractions were combined, evaporated under reduced pressure andco-evaporated with DCM. The residue was dried under vacuum at 50° C. toprovide Enantiomer 10A (164 mg). The product fractions of the secondeluted enantiomer were combined, evaporated under reduced pressure andpurified by flash chromatography on silica gel (12 g) using a gradientof heptane/EtOAc:EtOH:AcOH 100/0:0:0 to 60/30:9.8:0.2. The desiredfractions were combined, evaporated under reduced pressure andco-evaporated with DCM. The residue was dried under vacuum at 50° C. toprovide Enantiomer 10B (167 mg).

Compound 10:

¹H NMR (360 MHz, DMSO-d) 6 ppm 1.87 (t, J=6.8 Hz, 2H), 2.31-2.37 (m,2H), 3.26-3.38 (m, 2H), 3.62 (s, 3H), 3.84 (br t, J=6.4 Hz, 2H), 4.29(td, J=10.5, 6.8 Hz, 1H), 4.79 (td, J=10.2, 6.2 Hz, 1H), 5.49 (s, 1H),5.76 (t, J=2.0 Hz, 1H), 5.91-5.97 (m, 2H), 7.44 (d, J=8.4 Hz, 2H), 7.54(d, J=8.8 Hz, 2H), 7.76 (s, 1H)

LC/MS (method LC-D): R_(t) 1.93 min, MH⁺ 569

Enantiomer 10A:

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.83-1.91 (m, 2H), 2.30-2.36 (m, 2H),3.23-3.30 (m, 2H), 3.62 (br s, 3H), 3.85 (br s, 2H), 4.30 (m, J=9.5 Hz,1H), 4.79 (m, J=6.8 Hz, 1H), 5.48 (br d, J=8.8 Hz, 1H), 5.76 (br s, 1H),5.94 (br d, J=9.0 Hz, 2H), 6.35 (br d, J=8.1 Hz, 1H), 7.43 (br d, J=7.3Hz, 2H), 7.54 (br d, J=8.1 Hz, 2H), 7.76 (brs, 1H), 12.10 (brs, 1H)

LC/MS (method LC-C): R_(t) 1.03 min, MH⁺ 569

[α]_(D) ²⁰: +36.9° (c 0.4445, DMF)

Chiral SFC (method SFC-F): R_(t) 5.52 min, MH⁺ 569 chiral purity 100%.

Enantiomer 10B:

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.83-1.91 (m, 2H), 2.34 (br t, J=6.8 Hz,2H), 3.23-3.30 (m, 2H), 3.62 (s, 3H), 3.85 (br t, J=5.9 Hz, 2H),4.25-4.35 (m, 1H), 4.75-4.83 (m, 1H), 5.48 (br d, J=8.4 Hz, 1H), 5.76(br s, 1H), 5.94 (br d, J=8.8 Hz, 2H), 6.35 (br d, J=8.4 Hz, 1H), 7.43(br d, J=7.7 Hz, 2H), 7.54 (br d, J=7.9 Hz, 2H), 7.76 (s, 1H), 12.11 (brs, 1H)

LC/MS (method LC-C): R_(t) 1.03 min, MH⁺ 569

[α]_(D) ²⁰: −39.1 (c 0.437, DMF)

Chiral SFC (method SFC-F): R_(t) 6.98 min, MH⁺ 569 chiral purity 97%.

Example 11: Synthesis of4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-((trifluoromethyl)thio)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 11)

Synthesis of Intermediate 11a

To the suspension of NaH (26.5 g, 663 mmol, 60% in oil) in THF (100 mL)at 0° C. was added 6-bromo-1H-indole [CAS 52415-29-9] (100 g, 510 mmol)in portions. The reaction was stirred for 30 min at 15° C. After coolingto 0° C., SEMCl (93.6 g, 561 mmol, 99.5 mL) was added. The reactionmixture was stirred for 16 h at 15° C. and poured out into a saturatedaqueous ammonium chloride solution (200 mL). The mixture was dilutedwith ethyl acetate (300 mL). The layers were separated and the aqueouslayer was extracted with ethyl acetate (2×200 mL). The combined organiclayers were washed with brine (500 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel using petroleum ether. The product fractions were combinedand evaporated under reduced pressure to afford6-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole 11a (134 g) as ayellow oil.

Synthesis of Intermediate 11b

A mixture of 6-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole 11a(134 g, 411 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (158.5 g,624 mmol), Pd(dppf)Cl₂ (15.02 g, 20.5 mmol) and KOAc (161.2 g, 1.64 mol)in 1,4-dioxane (1.5 L) was stirred at 100° C. for 5 h underN₂-atmosphere. The reaction was cooled to 25° C. and filtered through apad of Celite®. The solvent was evaporated under reduced pressure andthe residue was purified by column chromatography on silica gel(petroleum ether/ethyl acetate gradient 100/0 to 50/1). The productfractions were combined and evaporated under reduced pressure to afford6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole11b (104 g) as a light yellow oil.

Synthesis of Intermediate 11c

To a solution of6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole11b (52 g, 139 mmol) in acetone (2.4 L) and H₂O (2.4 L) were added NaIO₄(119 g, 557 mmol) and NH₄OAc (53.7 g, 696 mmol). The reaction mixturewas stirred at 25° C. for 16 hours. The reaction was duplicated at thesame scale (52 g of compound 11b) and the reaction mixtures of bothreactions were combined for the work-up. The precipitate was filteredoff and the solvent (acetone) was removed under reduced pressure. Ethylacetate (5 L) was added and the organic layer was separated. The aqueouslayer was extracted with ethyl acetate (3×5 L). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated in vacuo toafford (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indol-6-yl)boronic acid11c (85 g) as a black brown solid which was used into the next stepwithout further purification.

Synthesis of Intermediate 11d

A mixture of TMSCF₃ (207.5 g, 1.46 mol), CuSCN (10.7 g, 87.6 mmol), S₈(224.6 g, 875.6 mmol),(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indol-6-yl)boronic acid 11c (85g, 292 mmol), Ag₂CO₃ (161 g, 584 mmol), K₃PO₄ (186 g, 876 mmol),1,10-phenanthroline (31.6 g, 175 mmol) and 4A molecular sieves (85 g) inDMF (1 L) was stirred at 25° C. for 16 hours under N₂-atmosphere. Thereaction mixture was filtered through a pad Celite®. The filtrate wasdiluted with MTBE (1 L), washed with water (3×500 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (petroleumether/ethyl acetate 100/1). The product fractions were combined andevaporated under reduced pressure to afford6-((trifluoromethyl)thio)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole11d (38 g) as an light yellow oil.

Synthesis of Intermediate 11e

To the solution of6-((trifluoromethyl)thio)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indole11d (38 g, 109 mmol) in THF (1.5 L) were added TBAF.3H₂O (345 g, 1.09mol) and ethane-1,2-diamine (131.45 g, 2.19 mol). The reaction mixturewas stirred at 70° C. for 16 h. The reaction mixture was cooled to 25°C. and poured out into saturated aqueous NaHCO₃ (3 L). The aqueousmixture was extracted with ethyl acetate (3×1 L). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by preparative HPLC (column:Phenomenex Gemini C18 250×50 mm 10 μm, mobile phase: water (0.05%ammonia hydroxide v/v), CH₃CN) to give6-((trifluoromethyl)thio)-1H-indole 11e (10.1 g) as an off-white solid.

Synthesis of Intermediate 11f

A mixture of 6-((trifluoromethyl)thio)-1H-indole 11e (1.0 g, 4.6 mmol)and borane dimethyl sulfide complex (7 mL) was heated in a sealed tubeat 75° C. for 5 h. The reaction mixture was allowed to reach roomtemperature and added dropwise to stirring MeOH (30 mL) (exothermic).After addition, the resulting solution was heated under reflux for 3 h.The solvent were evaporated under reduced pressure and the residue waspurified by flash chromatography on silica gel (25 g) using a gradientof heptane/CH₂Cl₂ 100/0 to 40/60. The desired fractions were combined,evaporated under reduced pressure, and co-evaporated with dioxane. Theproduct was dried under vacuum at 50° C. to provide6-((trifluoromethyl)thio)indoline 11f (0.79 g).

Synthesis of Intermediate 11g

A solution of 6-((trifluoromethyl)thio)indoline 11f (0.79 g, 3.6 mmol)in CH₃CN (30 mL) was stirred under N₂-atmosphere. NaHCO₃ (0.333 g, 3.96mmol) was added and the reaction mixture was cooled on an ice-bath. Asolution of 2-(4-chlorophenyl)acetyl chloride ([CAS 25026-34-0] (0.852g, 4.51 mmol) in CH₃CN (20 mL) was added, and the reaction mixture wasstirred at room temperature for 16 h. The mixture was poured out intostirring H₂O (100 mL). The precipitate was filtered off and washed withwater (4×10 mL). The solids were stirred up in Et₂O/heptane (3/2) (20mL), filtered off, washed with Et₂O/heptane (3/2) (2×10 mL) and driedunder vacuum at 50° C. to provide2-(4-chlorophenyl)-1-(6-((trifluoromethyl)thio)indolin-1-yl)ethanone 11g(1.033 g).

Synthesis of Intermediate 11h

At −78° C., under a N₂ flow, LiHMDS 1M in THF (5.56 mL, 5.56 mmol) wasadded dropwise to a mixture of2-(4-chlorophenyl)-1-(6-((trifluoromethyl)thio)indolin-1-yl)ethanone 11g(1.033 mg, 2.78 mmol) in 2-Me-THF (40 mL) and the mixture was kept at−78° C. for 20 min. TMSCl (568 μL, 4.45 mmol) was added dropwise. Themixture was stirred for 35 min at −78° C. and a solution ofN-bromosuccinimide (643 mg, 3.61 mmol) in THF (8 mL) was added dropwise.After stirring for 35 min at −78° C., the reaction was quenched with asaturated aqueous solution of NH₄Cl (30 mL). The cooling bath wasremoved and the reaction mixture was stirred until the reaction reachedroom temperature. Water (30 mL) and DIPE (30 mL) were added and themixture was stirred for 20 min. The organic layer was separated, washedwith brine, dried over MgSO₄, filtered and the solvent was evaporatedunder reduced pressure to give2-bromo-2-(4-chlorophenyl)-1-(6-((trifluoromethyl)thio)indolin-1-yl)ethanone11h (1.25 g), which was used as such in the next step.

Synthesis of Intermediate 11i

A mixture of2-bromo-2-(4-chlorophenyl)-1-(6-((trifluoromethyl)thio)indolin-1-yl)ethanone11h (1.25 mg, 2.78 mmol), tert-butyl4-(3-amino-5-methoxyphenoxy)butanoate 1a (1.56 g, 5.56 mmol) anddiisopropylethylamine (957 μL, 5.56 mmol) in 2-butanol (25 mL) wasstirred at 45° C. for 16 h. The reaction mixture was allowed to reachroom temperature, and poured out into stirring water (100 mL). Theproduct was extracted (2×) with CH₂Cl₂. The combined organic layers werewashed with brine, dried over MgSO₄, filtered, and the solvent wasevaporated under reduced pressure. The residue was purified by flashchromatography on silica gel (40 g) using a gradient ofheptane/EtOAc/EtOH 100/0/0 to 70/20/10. The desired fractions werecombined and evaporated under reduced pressure to provide tert-butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-((trifluoromethyl)thio)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate11i (2.0 g), which was used as such in the next step.

Synthesis of Compound 11

tert-Butyl4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-((trifluoromethyl)thio)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoate11i (1.81 g, 2.78 mmol), was mixed with 4M HCl in dioxane (20 mL) andthe mixture was stirred at room temperature for 3.5 h. The solids werefiltered off, washed with dioxane (3×) and Et₂O (20 mL). The solid wasdissolved in CH₂Cl₂ (100 mL) and mixed with water (50 mL) and saturatedaqueous Na₂CO₃ (30 mL). After stirring for 15 min, the layers wereseparated. The organic layer was washed with brine, dried over MgSO₄,filtered and evaporated under reduced pressure. The residue was purifiedvia preparative HPLC (Stationary phase: RP XBridge® Prep C18 OBD—10 μm,30×150 mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN). CH₃CNwas evaporated and the residual aqueous solution was acidified to pH 3with 1N HCl. The product was extracted with EtOAc (100 mL). The organiclayer was washed with brine (50 mL), dried over MgSO₄, filtered,evaporated under reduced pressure and co-evaporated with CH₂Cl₂ to give4-(3-((1-(4-chlorophenyl)-2-oxo-2-(6-((trifluoromethyl)thio)indolin-1-yl)ethyl)amino)-5-methoxyphenoxy)butanoicacid (Compound 11, 164 mg).

Compound 11:

¹H NMR (360 MHz, DMSO-d₆) δ ppm 1.87 (quin, J=7.1 Hz, 2H) 2.25-2.38 (m,2H) 3.16-3.27 (m, 2H) 3.62 (s, 3H) 3.81-3.87 (m, 2H) 3.95-4.08 (m, 1H)4.44-4.56 (m, 1H) 5.57 (br d, J=8.8 Hz, 1H) 5.74-5.77 (m, 1H) 5.90-5.98(m, 2H) 6.47 (br d, J=8.8 Hz, 1H) 7.34-7.40 (m, 2H) 7.41-7.48 (m, 2H)7.51-7.59 (m, 2H) 8.39 (s, 1H) 12.16 (br s, 1H)

LC/MS (method LC-C): R_(t) 1.12 min, MH⁺ 595

TABLE compounds prepared as described above Compound Structure Opticalrotation  1

racemic  1A

[α]_(D) ²⁰ = −44.6°  1B

[α]_(D) ²⁰ = +46.0°  2

racemic  2A

[α]_(D) ²⁰ = −39.0°  2B

[α]_(D) ²⁰ = +47.1°  3

racemic  3A

[α]_(D) ²⁰ = −48.9°  3B

[α]_(D) ²⁰ = +47.8°  4

racemic  4A

[α]_(D) ²⁰ = −39.6°  4B

[α]_(D) ²⁰ = +43.7°  5

racemic  5A

[α]_(D) ²⁰ = −35.8°  5B

[α]_(D) ²⁰ = +52.8°  6

racemic  6A

[α]_(D) ²⁰ = −37.3°  6B

[α]_(D) ²⁰ = +32.7°  7A-D

[α]_(D) ²⁰ = +54.2°  7B-D

[α]_(D) ²⁰ = −50.1°  8

racemic  9

racemic 10

racemic 10A

[α]_(D) ²⁰ = +36.9° 10B

[α]_(D) ²⁰ = −39.1° 11

racemic

Antiviral Activity of the Compounds of the Invention

DENV-2 Antiviral Assay

The antiviral activity of all the compounds of the invention was testedagainst the DENV-2 16681 strain which was labeled with enhanced greenfluorescent protein (eGPF). The culture medium consists of minimalessential medium supplemented with 2% of heat-inactivated fetal calfserum, 0.04% gentamycin (50 mg/mL) and 2 mM of L-glutamine. Vero cells,obtained from ECACC, were suspended in culture medium and 25 μL wasadded to 384-well plates (2500 cells/well), which already contain theantiviral compounds. Typically, these plates contain a 5-fold serialdilution of 9 dilution steps of the test compound at 200 times the finalconcentration in 100% DMSO (200 nL). In addition, each compoundconcentration is tested in quadruplicate (final concentration range: 25μM-0.000064 μM or 2.5 μM-0.0000064 μM for the most active compounds).Finally, each plate contains wells which are assigned as virus controls(containing cells and virus in the absence of compound), cell controls(containing cells in the absence of virus and compound) and mediumcontrols (containing medium in the absence of cells, virus andcompounds). To the wells assigned as medium control, 25 μL of culturemedium was added instead of Vero cells. Once the cells are added to theplates, the plates were incubated for 30 minutes at room temperature toallow the cells to distribute evenly within the wells. Next, the plateswere incubated in a fully humidified incubator (37° C., 5% CO₂) untilthe next day. Then, DENV-2 strain 16681, labeled with eGFP, was added ata multiplicity of infection (MOI) of 0.5.

Therefore, 15 μL of virus suspension was added to all the wellscontaining test compound and to the wells assigned as virus control. Inparallel, 15 μL of culture medium was added to the medium and cellcontrols. Next, the plates were incubated for 3 days in a fullyhumidified incubator (37° C., 5% CO₂). At the day of the read out, theeGFP fluorescence was measured using an automated fluorescencemicroscope at 488 nm (blue laser). Using an in-house LIMS system,inhibition dose response curves for each compound were calculated andthe half maximal effective concentration (EC₅₀) was determined.Therefore, the percent inhibition (I) for every test concentration iscalculated using the following formula:I=100*(S_(T)−S_(CC))/(S_(VC)−S_(CC)); S_(T), S_(CC) and S_(VC) are theamount of eGFP signal in the test compound, cell control and viruscontrol wells, respectively. The EC₅₀ represents the concentration of acompound at which the virus replication is inhibited with 50%, asmeasured by a 50% reduction of the eGFP fluorescent intensity comparedto the virus control. The EC₅₀ is calculated using linear interpolation(Table 1).

In parallel, the toxicity of the compounds was assessed on the sameplates. Once the read-out for the eGFP signal was done, 40 μL ofATPlite, a cell viability stain, was added to all wells of the 384-wellplates. ATP is present in all metabolically active cells and theconcentration declines very rapidly when the cells undergo necrosis orapoptosis. The ATPLite assay system is based on the production of lightcaused by the reaction of ATP with added luciferase and D-luciferin. Theplates were incubated for 10 minutes at room temperature. Next, theplates were measured on a ViewLux. The half maximal cytotoxicconcentration (CC₅₀) was also determined, defined as the concentrationrequired to reduce the luminescent signal by 50% compared to that of thecell control wells. Finally, the selectivity index (SI) was determinedfor the compounds, which was calculated as followed: SI=CC₅₀/EC₅₀.

TABLE 1 EC₅₀, CC₅₀, and SI for the compounds of the invention in theDENV-2 antiviral assay compound # EC₅₀ (μM) N CC₅₀ (μM) N SI N  10.00064 3 13 4 19800 3  1A 0.0013 3 12 3 9200 3  1B 0.00011 3 13 4104092 3  2 0.00038 3 12 4 32100 3  2A 0.015 3 12 3 799 3  2B 0.000078 414 4 166670 4  3 0.00056 3 13 3 22700 3  3A 0.036 3 12 3 346 3  3B0.00012 3 13 3 91000 3  4 0.00011 4 12 4 96000 4  4A 0.011 3 13 3 1180 3 4B 0.000057 4 13 4 186421 4  5 0.00011 3 10 3 90900 3  5A 0.0023 6 10 94440 6  5B 0.00012 4 12 4 >54214 4  6 0.00063 3 12 3 19500 3  6A 0.25 311 3 46 3  6B 0.00039 3 15 4 39700 3  7A-D 0.000100 3 12 3 118813 3 7B-D 0.016 3 9.6 3 584 3  8 0.00015 3 13 4 86800 3  9 0.00099 4 12 412600 4 10 0.00052 3 19 3 40900 3 10A 0.00030 3 14 3 58900 3 10B 0.037 312 3 330 3 11 0.00028 3 13 3 43300 3 N = the number of independentexperiments in which the compounds were tested.

Tetravalent Reverse Transcriptase Quantitative-PCR (RT-qPCR) Assay

The antiviral activity of the compounds of the invention was testedagainst DENV-1 strain TC974#666 (NCPV), DENV-2 strain 16681, DENV-3strain H87 (NCPV) and DENV-4 strain H241 (NCPV) in a RT-qPCR assay.Therefore, Vero cells were infected with either DENV-1, or -2, or -3, or-4 in the presence or absence of test compounds. At day 3post-infection, the cells were lysed and cell lysates were used toprepare cDNA of both a viral target (the 3′UTR of DENV; Table 2) and acellular reference gene (1-actin, Table 2). Subsequently, a duplex realtime PCR was performed on a Lightcycler480 instrument. The generated Cpvalue is inversely proportional to the amount of RNA expression of thesetargets. Inhibition of DENV replication by a test compound results in ashift of Cp's for the 3′UTR gene. On the other hand, if a test compoundis toxic to the cells, a similar effect on 1-actin expression will beobserved. The comparative ΔΔCp method is used to calculate EC₅₀, whichis based on the relative gene expression of the target gene (3′UTR)normalized with the cellular housekeeping gene (1-actin). In addition,CC₅₀ values are determined based on the C, values acquired for thehousekeeping gene β-actin.

TABLE 2 Primers and probes used for the real-time, quantitative RT-PCR.Primer/ probe Target Sequence^(a, b) F3utr258 DENV 5′-CGGTTAGAGGAGACCC3′-UTR CTC-3′ R3utr425 DENV 5′-GAGACAGCAGGATCTC 3′-UTR TGGTC-3′ P3utr343DENV

5′-AAGGACTAG- 3′-UTR ZEN-AGGTTAGAGGAGACC CCCC-3′-

Factin743 β-actin 5′-GGCCAGGTCATCACCA TT-3′ Ractin876 β-actin5′-ATGTCCACGTCACACT TCATG-3′ Pactin773 β-actin

5′-TTCCGCTGC-

CCTGAGGCTCT C-3′-

^(a)Reporter dyes (FAM, HEX) and quenchers (ZEN and lABkFQ) elements areindicated in bold and italics. ^(b)The nucleotide sequence of theprimers and probes were selected from the conserved region in the 3′UTRregion of the dengue virus genome, based on the alignment of 300nucleotide sequences of the four dengue serotypes deposited in Genbank(Gong et al., 2013, Methods Mol Biol, Chapter 16).

The culture medium consisted of minimal essential medium supplementedwith 2% of heat-inactivated fetal calf serum, 0.04% gentamycin (50mg/mL) and 2 mM of L-glutamine. Vero cells, obtained from ECACC, weresuspended in culture medium and 75 μL/well was added in 96-well plates(10000 cells/well), which already contain the antiviral compounds.Typically, these plates contain a 5-fold serial dilution of 9 dilutionsteps of the test compound at 200 times the final concentration in 100%DMSO (500 nL; final concentration range: 25 μM-0.000064 μM or 2.5μM-0.0000064 μM for the most active compounds). In addition, each platecontains wells which are assigned as virus controls (containing cellsand virus in the absence of compound) and cell controls (containingcells in the absence of virus and compound). Once the cells were addedin the plates, the plates were incubated in a fully humidified incubator(37° C., 5% CO₂) until the next day. Dengue viruses serotype-1, 2, 3 and4 were diluted in order to obtain a Cp of ˜22-24 in the assay.Therefore, 25 μL of virus suspension was added to all the wellscontaining test compound and to the wells assigned as virus control. Inparallel, 25 μL of culture medium was added to the cell controls. Next,the plates were incubated for 3 days in a fully humidified incubator(37° C., 5% CO₂). After 3 days, the supernatant was removed from thewells and the cells were washed twice with ice-cold PBS (−100 μL). Thecell pellets within the 96-well plates were stored at −80° C. for atleast 1 day. Next, RNA was extracted using the Cells-to-CT™ lysis kit,according to the manufacturer's guideline (Life Technologies). The celllysates can be stored at −80° C. or immediately used in the reversetranscription step.

In preparation of the reverse transcription step, mix A (table 3A) wasprepared and 7.57 μL/well was dispensed in a 96-well plate. Afteraddition of 5 μL of the cell lysates, a five minute denaturation step at75° C. was performed (table 3B). Afterwards, 7.43 μL of mix B was added(table 3C) and the reverse transcription step was initiated (table 3D)to generate cDNA.

Finally, a RT-qPCR mix was prepared, mix C (table 4A), and 22.02 μL/wellwas dispensed in 96-well LightCycler qPCR plates to which 3 μL of cDNAwas added and the qPCR was performed according to the conditions intable 4B on a LightCycler 480.

Using the LightCycler software and an in-house LIMS system, doseresponse curves for each compound were calculated and the half maximaleffective concentration (EC₅₀) and the half maximal cytotoxicconcentration (CC₅₀) were determined (Tables 5-8).

TABLE 3 cDNA synthesis using Mix A, denaturation, Mix B and reversetranscription. A Mix A Plates  8 Samples 828 Reaction Vol. (μl) 20Concentration Volume for (μl) Mix Item Unit Stock Final 1 sample xsamples Milli-Q H₂O 7.27 6019.56 R3utr425 μM 20 0.27 0.15  124.20Ractin876 μM 20 0.27 0.15  124.20 Volume mix/well (μl) 7.57 Cell lysates5.00 B Denaturation step: Step Temp Time Denaturation 75° C. 5′ Hold  4°C. hold C Mix B Samples 864 Concentration Volume for (μl) Mix Item UnitStock Final 1 sample x samples Expand HIFI X 10.00 1.00 2.00 1728.0buffer 2 MgCl₂ mM 25.00 3.50 2.80 2419.2 dNTPs mM 10.00 1.00 2.00 1728.0Rnase inhibitor U/μl 40.00 1.00 0.50  432.0 Expand RT U/μl 50.00 0.330.13  112.3 Total Volume Mix (μl) 7.43 D Protocol cDNA synthesis StepTemp Time Rev transc 42° C. 30′ Denaturation 99° C.  5′ Hold  4° C. hold

TABLE 4 qPCR mix and protocol. A mix c Samples 833 Reaction Vol. (μl) 25Concentration Volume for (μl) Mix Item Unit Stock Final 1 sample xsamples H₂O PCR grade Roche  7.74  6447.42 Roche 2 × MM mix X  2   112.50 10412.50 F3utr258 μM 20 0.3  0.38  316.54 R3utr425 μM 20 0.3  0.38 316.54 P3utr343 μM 20 0.1  0.13  108.29 Factin743 μM 20 0.3  0.38 316.54 Ractin876 μM 20 0.3  0.38  316.54 Pactin773 μM 20 0.1  0.13 108.29 Volume Mix/Tube (μl) 22.02 cDNA  3.00 B Protocol qPCR3 Step TempTime Ramp rate preincub/denat 95° C. 10 min 4.4 Denaturation 95° C. 10sec 4.4 40 cycles annealing 58° C. 1 min 2.2 Elongation 72° C. 1 sec 4.4Cooling 40° C. 10 sec 1.5

TABLE 5 EC₅₀, CC₅₀, and SI for the compounds against serotype 1 in theRT-qPCR assays RT-qPCR serotype 1 TC974#666 compound EC50 CC50 # (μM) N(μM) N SI N  1B 0.000096 4 >2.5 4 >79500 4  2B 0.000091 5 >1.0 5 >331005  3B 0.00010 3 >2.5 3 >54200 3  4B 0.00011 4 >1.0 4 >45200 4  5B0.00033 3 >1.0 3 >5910 3  6B 0.00064 4 13 4 20500 4  7A-D 0.00024 3 >1.03 >6180 3 10A 0.00022 5 13 5 56000 5 N = the number of independentexperiments in which the compounds were tested.

TABLE 6 EC₅₀, CC₅₀, and SI for the compounds against serotype 2 in theRT-qPCR assays RT-qPCR serotype 2 16681 compound EC₅₀ CC₅₀ # (μM) N (μM)N SI N  1B 0.00018 4 >2.5 4 >11700 4  2B 0.000061 4 >1.0 4 >36300 4  3B0.000096 3 >2.5 3 >46900 3  4B 0.000067 4 >1.0 4 >39400 4  5B 0.000293 >1.0 3 >5770 3  6B 0.00041 3 15 4 28100 3  7A-D 0.00016 3 >1.0 3 >93303 10A 0.00011 6 15 5 131977 5 N = the number of independent experimentsin which the compounds were tested.

TABLE 7 EC₅₀, CC₅₀, and SI for the compounds against serotype 3 in theRT-qPCR assays RT-qPCR serotype 3 H87 compound EC₅₀ CC₅₀ # (μM) N (μM) NSI N  1B 0.0019 4 >2.5 4 >1590 4  2B 0.00085 4 >1.0 4 >2050 4  3B 0.00153 >2.5 3 >3870 3  4B 0.00092 4 >1.0 4 >2360 4  5B 0.0026 3 >1.0 3 >719 3 6B 0.0056 4 13 4 2520 4  7A-D 0.0024 3 >1.0 3 >574 3 10A 0.0042 5 15 56210 5 N = the number of independent experiments in which the compoundswere tested.

TABLE 8 EC₅₀, CC₅₀, and SI for the compounds against serotype 4 in theRT-qPCR assays RT-q PCR serotype 4 H241 compound EC₅₀ CC₅₀ # (μM) N (μM)N SI N  1B 0.0096 4 8.8 4 2980 4  2B 0.010 4 4.1 4 1020 4  3B 0.014 33.6 1 333 1  4B 0.012 3 6.8 2 563 2  5B 0.020 3 8.4 3 618 3  6B 0.029 49.7 3 317 3  7A-D 0.013 3 8.2 3 1000 3 10A 0.030 5 3.2 5 105 5 N = thenumber of independent experiments in which the compounds were tested.

The invention claimed is:
 1. A compound of formula (I), including anystereochemically isomeric form thereof,

wherein A is

wherein R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ ispentafluorosulfanyl, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen; orR¹ is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethyl, R⁵ is hydrogen, Z is carbon, and R⁶ is methyl; or R¹ ischloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethyl, R⁵ is fluoro, Z is carbon, and R⁶ is hydrogen; or R¹ ischloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is methyl; or R¹is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethoxy, R⁵ is fluoro, Z is carbon, and R⁶ is hydrogen; or R¹is chloro, R² is hydrogen, R³ is deuterium, A is (a-1), R⁴ istrifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen; or R¹is chloro, R² is —OCH₂CH₂OH, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethoxy, R⁵ is hydrogen, Z is carbon, and R⁶ is hydrogen; or R¹is chloro, R² is hydrogen, R³ is hydrogen, A is (a-1), R⁴ istrifluoromethyl, R⁵ is methoxy, Z is nitrogen, and R⁶ is absent; or R¹is chloro, R² is hydrogen, R³ is hydrogen, A is (a-2), and R⁴ istrifluoromethyl; or R¹ is chloro, R² is hydrogen, R³ is hydrogen, A is(a-1), R⁴ is trifluoromethylthio, R⁵ is hydrogen, Z is carbon, and R⁶ ishydrogen; or a pharmaceutically acceptable salt, solvate or polymorphthereof.
 2. The compound according to claim 1 wherein A is (a-1).
 3. Thecompound according to claim 1 wherein A is (a-2).
 4. The compoundaccording to claim 1 wherein said compound has the (+) specificrotation.
 5. The compound according to claim 1 wherein said compound isselected from:


6. A pharmaceutical composition comprising the compound according toclaim 1 together with one or more pharmaceutically acceptableexcipients, diluents or carriers.
 7. The pharmaceutical compositionaccording to claim 6 which comprises a second or further activeingredient.
 8. The pharmaceutical composition according to claim 7wherein the second or further active ingredient is an antiviral agent.9. A method of treating or preventing Dengue infection or a diseasecaused by Dengue infection, comprising administering to a subject inneed thereof an effective amount of the compound according to claim 1.10. The method according to claim 9 wherein the Dengue infection is aninfection by viruses of one of the DENV-1, DENV-2, DENV-3 or DENV-4strain.
 11. The method according to claim 9, wherein the methodcomprises treating Dengue infection or a disease caused by Dengueinfection, wherein said disease is Dengue fever, Dengue hemorrhagicfever or Dengue shock syndrome.
 12. The method according to claim 9,wherein the method comprises preventing Dengue infection or a diseasecaused by Dengue infection, wherein said disease is Dengue fever, Denguehemorrhagic fever or Dengue shock syndrome.
 13. A method of treating orpreventing Dengue infection or a disease caused by Dengue infectioncomprising administering to a subject in need thereof an effectiveamount of the pharmaceutical composition of claim
 6. 14. The methodaccording to claim 13, wherein the method comprises treating Dengueinfection or a disease caused by Dengue infection, wherein said diseaseis Dengue fever, Dengue hemorrhagic fever or Dengue shock syndrome. 15.The method according to claim 13, wherein the method comprisespreventing Dengue infection or a disease caused by Dengue infection,wherein said disease is Dengue fever, Dengue hemorrhagic fever or Dengueshock syndrome.
 16. A method of inhibiting Dengue virus replication inan animal cell, comprising administering to a subject in need thereof aneffective amount of the compound according to claim
 1. 17. A method ofinhibiting Dengue virus replication in an animal cell, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition according to claim
 6. 18. The compoundaccording to claim 5 wherein said compound is


19. The compound according to claim 5 wherein said compound is


20. The compound according to claim 5 wherein said compound is


21. The compound according to claim 5 wherein said compound is


22. The compound according to claim 5 wherein said compound is


23. The compound according to claim 5 wherein said compound is


24. The compound according to claim 5 wherein said compound is


25. The method according to claim 13 wherein the Dengue infection is aninfection by viruses of one of the DENV-1, DENV-2, DENV-3 or DENV-4strain.